Compositions and methods for ameliorating CNS inflammation, psychosis, delirium, PTSD or PTSS

ABSTRACT

The invention provides compositions and methods for ameliorating, treating, reversing or preventing pathology or inflammation in the central nervous system (CNS), or the brain, caused or mediated by NFkB, IL-6, IL-6-R, NADPH oxidase (Nox), and/or superoxide and/or hydrogen peroxide production by a NADPH oxidase, including for example ameliorating, treating, reversing or preventing schizophrenia, psychosis, delirium, e.g., post-operative delirium, drug-induced psychosis, psychotic features associated with frailty syndrome (FS), aging, depression, dementias; traumatic war neurosis, post traumatic stress disorder (PTSD) or post-traumatic stress syndrome (PTSS), Amyotrophic Lateral Sclerosis (ALS, or Lou Gehrig&#39;s Disease), and/or Multiple Sclerosis (MS). The invention also provides methods for purifying a C60 fullerene, C 3  (tris malonic acid C60) or malonic acid derivatives.

RELATED APPLICATIONS

This Patent Convention Treaty (PCT) International Application claimsbenefit of priority to U.S. Provisional Patent Application Ser. No.60/999,587, filed Oct. 19, 2007. The aforementioned application isexpressly incorporated herein by reference in its entirety and for allpurposes.

TECHNICAL FIELD

This invention relates to molecular and cellular biology, biochemistryand medicine. The invention provides compositions and methods forameliorating or preventing pathologies or inflammation in the centralnervous system (CNS), or the brain, caused or mediated by NFkB,interleukin-6 (IL-6), NADPH oxidase (“Nox”), superoxide dismutase (SOD),and/or superoxide and/or hydrogen peroxide production by an NADPHoxidase, including e.g., ameliorating or preventing schizophrenia,psychosis, delirium, drug-induced psychosis, psychotic featuresassociated with these conditions, frailty syndrome (FS), cognitive,learning or memory impairments associated with frailty syndrome (FS),aging, depression and/or dementias; traumatic war neurosis, posttraumatic stress disorder (PTSD) and/or post-traumatic stress syndrome(PTSS), and/or Amyotrophic Lateral Sclerosis (ALS, or Lou Gehrig'sDisease) and/or Multiple Sclerosis (MS); and cognitive, learning ormemory impairments resulting therefrom. The invention also providesmethods for purifying a C60 fullerene, C₃ (tris malonic acid C60) or amalonic acid derivatives.

BACKGROUND

Schizophrenia, psychosis, delirium, drug-induced psychosis, psychoticfeatures associated with depression and dementia, and dementias areincreasingly prevalent and important medical condition. Although theneural circuitry changes that are believed to be responsible for thesedeficits have been well described in humans, and are reproduced inprimate and rodent models of these same disorders, there are currentlyno therapies directed at the underlying causes of these neural circuitrychanges.

Interleukin-6 (IL-6) is known to be elevated in patients with psychosis,schizophrenia, and many dementing disorders. Recently, a therapeutichumanized monoclonal antibody (tocilizumab, or ACTEMRA™ (F. Hoffmann-LaRoche Ltd, Basel, Switzerland)) acting as a specific antagonist (isreceptor-inhibiting) for IL-6 receptors was approved for the treatmentof arthritis.

Frailty syndrome (FS) has become increasingly recognized as a majorpredictor of co-morbidities and mortality in older individuals. Whiledefinitions of FS vary, most experts agree this syndrome ischaracterized by reduced functional reserve, impaired adaptive responsesresulting multi-system decline, which results in increased vulnerabilityto adverse events.

SUMMARY

The invention provides compositions and methods for preventing orameliorating an inflammation, pathology or condition in the centralnervous system, e.g., the brain, caused or mediated by NFkB,interleukin-6 (IL-6), interleukin-6 (IL-6) receptor (IL-6-R) and/or anymember of the NADPH oxidase enzyme family (collectively referred to as“Nox”; e.g., Nox1, Nox2, Nox3, Nox4 or Nox5) or the superoxide dismutase(SOD) enzyme family, and/or superoxide and/or hydrogen peroxideproduction by a NADPH oxidase, including preventing or ameliorating e.g.schizophrenia, psychosis, delirium, e.g., post-operative delirium,drug-induced psychosis; psychotic features, frailty syndrome (FS),cognitive, learning or memory impairments associated with frailtysyndrome (FS), aging, depression and/or dementias (e.g., fromAlzheimer's disease, Lewy Body Disease, Parkinson's Disease,Huntington's Disease, Multi-infarct dementia, senile dementia orFrontotemporal Dementia); preventing or ameliorating Amyotrophic LateralSclerosis (ALS, or Lou Gehrig's Disease). Multiple Sclerosis (MS),traumatic war neurosis, post traumatic stress disorder (PTSD) orpost-traumatic stress syndrome (PTSS), and cognitive, learning or memoryimpairments resulting therefrom, frailty syndrome (FS), aging,inflammation from CNS infections such as bacterial, yeast or viralinfections, e.g., HIV infection (e.g., HIV-1 infection, or AIDS) ormeningitis, including Haemophilus, Cryptococcus, Filobasidiella,Neisseria, Rickettsia or Borrelia infections, and the like. Thecompositions and methods of this invention can be used to inhibit theactivity of or decrease levels of superoxide and/or hydrogen peroxideproduction by inhibiting or decreasing the activity of NFkB, IL-6,IL-6-R and/or the enzyme NADPH oxidase. In one embodiment, compositionsof the invention (e.g., superoxide dismutase (SOD) mimetics) and methodsof this invention are used as superoxide dismutase (SOD) mimetics (tomimic the activity of SOD) to decrease levels of superoxide and/orhydrogen peroxide production.

The invention provides methods for ameliorating or preventingschizophrenia, psychosis, delirium, e.g., post-operative delirium,drug-induced psychosis, psychotic features, frailty syndrome (FS), orcognitive, learning or memory impairments resulting from or associatedwith frailty syndrome (FS), aging, depression, dementias; amelioratingor preventing Amyotrophic Lateral Sclerosis (ALS, or Lou Gehrig'sDisease), Multiple Sclerosis (MS), trauma, traumatic war neurosis, posttraumatic stress disorder (PTSD), post-traumatic stress syndrome (PTSS),and cognitive, learning or memory impairments resulting therefrom, andinflammation from CNS infections in an individual comprising:

(a) (i) providing a composition that

(1) inhibits or decreases the in vivo activity of NFkB, interleukin-6(IL-6), interleukin-6 receptor (IL-6-R) or a member of the NADPH oxidaseenzyme family (Nox), and/or inhibits or decreases superoxide and/orhydrogen peroxide production by a member of the NADPH oxidase enzymefamily, or

(2) acts as a superoxide dismutase mimetic to decrease superoxide and/orhydrogen peroxide; and

(ii) administering an effective amount of the composition of (a) to theindividual in need thereof;

(b) the method of (a), wherein the individual is a human;

(c) the method of (a) or (b), wherein the composition comprises apharmaceutical formulation;

(d) the method of (c), wherein the pharmaceutical formulation isformulated for delivery to the brain or a neural cell, or for passingthrough the blood brain barrier (BBB);

(e) the method of (d), wherein the pharmaceutical formulation isformulated for delivery to a parvalbumin-positive GABA-ergicinterneuron;

(f) the method of any of (a) to (c), wherein the pharmaceuticalformulation comprises a therapeutic monoclonal antibody specific for andinhibitory to the activity of NFkB, an IL-6 or IL-6-R and/or an NADPHoxidase enzyme;

(g) the method of (f), wherein the therapeutic monoclonal antibody is ahumanized or a human antibody; or

(h) the method of (f) or (g), wherein the therapeutic monoclonalantibody against the IL-6-R is tocilizumab, or ACTEMRA™, or thetherapeutic monoclonal antibody is against IL-6, or is CNTO-328, ahuman-mouse chimeric monoclonal antibody (Mab) to IL-6;

(i) the method of (a) or (b), wherein the composition comprises a smallmolecule;

(j) the method of (i), wherein the small molecule comprises ano-methoxycatechol, an apocynin, a diapocynin,4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF),4-hydroxy-3′-methoxy-acetophenon, N-Vanillylnonanamide, staurosporine orrelated compounds;

(k) the method of (a) or (b), wherein the composition comprises aninhibitory nucleic acid molecule to NFkB, IL-6 or NADPH oxidase;

(l) the method of (k), wherein the inhibitory nucleic acid moleculecomprises an RNAi molecule, a double-stranded RNA (dsRNA) molecule, ansiRNA, a miRNA (microRNA) and/or a short hairpin RNA (shRNA) molecule,or a ribozyme, or a fragment of an NADPH oxidase-encoding nucleic acid;

(m) the method of any of (a) to (l), wherein the member of the NADPHoxidase enzyme family (Nox) is a Nox1, Nox2, Nox3, Nox4 or Nox5 enzyme;

(n) the method of any of (a) to (l), wherein the infection is a viral,bacterial, yeast and/or fungal infection, or a Haemophilus,Cryptococcus, Filobasidiella, Neisseria, Rickettsia or Borreliainfection:

(o) the method of any of (a) to (n), wherein the composition thatinhibits or decreases the in vivo activity of NFkB, interleukin-6 (IL-6)or IL-6R is an antibody against NFkB IL-6 or IL-6-R, respectively;

(p) the method of (o), wherein the composition that inhibits ordecreases the in vivo activity of interleukin-6 (IL-6) is interleukin-10(IL-10);

(q) the method of (p), wherein the composition that inhibits ordecreases the in vivo activity of interleukin-6 (IL-6) is ilodecakin orTENOVIL™; or

(r) the method of any of (a) to (n), wherein superoxide dismutasemimetic that decreases superoxide and/or hydrogen peroxide comprises aC60 fullerene, C₃ (tris malonic acid C60) or a malonic acid derivative.

The invention provides methods for protecting the function of, ormaintaining the level of activation or activity of cortical inhibitoryneurons, or parvalbumin-positive GABA-ergic interneurons, comprising:

(a) (i) providing a composition that

(1) inhibits or decreases the in vivo activity of NFkB, interleukin-6(IL-6), interleukin-6 receptor (IL-6-R) or a member of the NADPH oxidaseenzyme family (Nox), and/or inhibits or decreases superoxide and/orhydrogen peroxide production by a member of the NADPH oxidase enzymefamily, or

(2) acts as a superoxide dismutase mimetic to decrease superoxide and/orhydrogen peroxide; and

(ii) contacting the composition of (a) with the cortical inhibitoryneuron or parvalbumin-positive GABA-ergic interneuron;

(b) the method of (a), wherein the contacting is in vivo or in vitro:

(c) the method of (b), wherein the contacting is in vivo and thecomposition of (a) is administered in an effective amount to anindividual in need thereof;

(d) the method of (c), wherein the contacting is in vivo to the CNS, orbrain cortex, of the individual;

(e) the method of (c) or (d), wherein the individual is a human;

(f) the method of any of (a) to (e), wherein the composition comprises apharmaceutical formulation;

(g) the method of (f), wherein the pharmaceutical formulation isformulated for delivery to the brain or a neural cell, or for passingthrough the blood brain barrier (BBB);

(h) the method of (f) or (g), wherein the pharmaceutical formulation isformulated for delivery to a cortical inhibitory neuron or aparvalbumin-positive GABA-ergic interneuron;

(i) the method of any of (a) to (h), wherein the composition or thepharmaceutical formulation comprises a therapeutic monoclonal antibodyspecific for and inhibitory to the activity of NFkB, an IL-6 or IL-6-Ror an NADPH oxidase enzyme;

(j) the method of (i), wherein the therapeutic monoclonal antibody is ahumanized or a human antibody; or

(k) the method of (i) or (j), wherein the therapeutic monoclonalantibody against the IL-6-R is tocilizumab, or ACTEMRA™, or thetherapeutic monoclonal antibody is against IL-6, or is CNTO-328, ahuman-mouse chimeric monoclonal antibody (Mab) to IL-6;

(l) the method of any of (a) to (h), wherein the composition or thepharmaceutical formulation comprises a small molecule:

(m) the method of (l), wherein the small molecule comprises ano-methoxycatechol, an apocynin, a diapocynin,4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF),4-hydroxy-3′-methoxy-acetophenon, N-Vanillylnonanamide, staurosporine orrelated compounds;

(n) the method of any of (a) to (h), wherein the composition or thepharmaceutical formulation comprises an inhibitory nucleic acid moleculeto NFkB, IL-6, IL-6-R, or NADPH oxidase; or

(o) the method of (n), wherein the inhibitory nucleic acid moleculecomprises an RNAi molecule, a double-stranded RNA (dsRNA) molecule, ansiRNA, a miRNA (microRNA) and/or a short hairpin RNA (shRNA) molecule,or a ribozyme, or a fragment of an NADPH oxidase-encoding nucleic acid;or

(p) the method of any of (a) to (o), wherein the member of the NADPHoxidase enzyme family (Nox) is a Nox1, Nox2, Nox3, Nox4 or Nox5 enzyme;

(q) the method of any of (a) to (p), wherein the composition thatinhibits or decreases the in vivo activity of NFkB, IL-6, IL-6-R, orNADPH oxidase is an antibody against NFkB, IL-6, IL-6-R, or NADPHoxidase, respectively;

(r) the method of (q), wherein the composition that inhibits ordecreases the in vivo activity of interleukin-6 (IL-6) is IL-10;

(s) the method of (r), wherein the composition that inhibits ordecreases the in vivo activity of interleukin-6 (IL-6) is ilodecakin orTENOVIL™; or

(t) the method of any of (a) to (h), wherein superoxide dismutasemimetic that decreases superoxide and/or hydrogen peroxide comprises aC60 fullerene, C₃ (tris malonic acid C60) or a malonic acid derivative.

The invention provides kits comprising

(a) a composition that inhibits NFkB, interleukin-6 (IL-6),interleukin-6 receptor, a member of the NADPH oxidase enzyme family(Nox), and/or superoxide or hydrogen peroxide production by a member ofthe NADPH oxidase enzyme family (Nox), or a composition that acts as asuperoxide dismutase mimetic to decrease superoxide and/or hydrogenperoxide;

(b) the kit of (a) further comprising instructions comprising use of themethod of claim 1 or claim 2;

(c) the kit of any of (a) or (b), wherein the member of the NADPHoxidase enzyme family (Nox) is a Nox1, Nox2, Nox3, Nox4 or Nox5 enzyme;

(d) the kit of any of (a) to (c), wherein the composition that inhibitsNFkB, interleukin-6 (IL-6), interleukin-6 receptor, a member of theNADPH oxidase enzyme family (Nox), and/or superoxide and/or hydrogenperoxide production by a member of the NADPH oxidase enzyme family (Nox)comprises an antisense nucleic acid, an siRNA, a miRNA or a ribozymethat binds to hybridization to and inhibits or decreases the activity orexpression of an antibody the specifically binds to the NFkB,interleukin-6 (IL-6), interleukin-6 receptor, or the member of the NADPHoxidase enzyme family (Nox); or

(e) the kit of any of (a) to (c), wherein the composition that inhibitsthe NFkB, interleukin-6 (IL-6), interleukin-6 receptor, or the member ofthe NADPH oxidase enzyme family (Nox), and/or superoxide and/or hydrogenperoxide production by a member of the NADPH oxidase enzyme family (Nox)comprises an antibody that specifically binds to the NFkB, interleukin-6(IL-6), interleukin-6 receptor, or the member of the NADPH oxidaseenzyme family (Nox), respectively;

(f) the kit of any of (a) to (e), wherein the composition that inhibitsor decreases the in vivo activity of interleukin-6 (IL-6) is an antibodyagainst IL-6;

(g) the kit of (f), wherein the composition that inhibits or decreasesthe in vivo activity of interleukin-6 (IL-6) is IL-10;

(h) the kit of (g), wherein the composition that inhibits or decreasesthe in vivo activity of interleukin-6 (IL-6) is ilodecakin or TENOVIL™;or

(i) the kit of (a), wherein superoxide dismutase mimetic that decreasessuperoxide and/or hydrogen peroxide comprises a C60 fullerene, C₃ (trismalonic acid C60) or a malonic acid derivative.

The invention provides uses of a composition that inhibits or decreasesthe level of activation or activity of NFkB, interleukin-6 (IL-6),interleukin-6 receptor (IL-6-R) or a member of the NADPH oxidase enzymefamily (Nox), and/or superoxide or hydrogen peroxide production by amember of the NADPH oxidase enzyme family (Nox), for the manufacture ofa pharmaceutical for protecting the function of, or maintaining thelevel of activation or activity of, a parvalbumin-positive GABA-ergicinterneuron in the cortex of an individual. In one embodiment of theuse: (a) the member of the NADPH oxidase enzyme family (Nox) is a Nox1,Nox2, Nox3, Nox4 or Nox5 enzyme, (b) the composition that inhibits NFkB,interleukin-6 (IL-6), interleukin-6 receptor (IL-6-R) or a member of theNADPH oxidase enzyme family (Nox), comprises an antisense nucleic acid,an siRNA, a miRNA or a ribozyme that binds to or hybridizes to andinhibits or decreases the activity or expression of a nucleic acidencoding NFkB, interleukin-6 (IL-6), interleukin-6 receptor (IL-6-R) ora member of the NADPH oxidase enzyme family (Nox). In one embodiment ofthe use, the composition that inhibits NFkB, interleukin-6 (IL-6),interleukin-6 receptor (IL-6-R) or a member of the NADPH oxidase enzymefamily (Nox) is an antibody the specifically binds to the NFkB, IL-6,IL-6-R or the member of the NADPH oxidase enzyme family (Nox).

The invention provides uses of a composition that acts as a superoxidedismutase mimetic to decrease superoxide and/or hydrogen peroxide,wherein in one embodiment the superoxide dismutase mimetic thatdecreases superoxide and/or hydrogen peroxide comprises a C60 fullerene,C₃ (tris malonic acid C60) or a malonic acid derivative.

The invention provides uses of a composition that inhibits or decreasesthe level of activation or activity of NFkB, IL-6, IL-6-R, NADPHoxidase, and/or decreases the level of superoxide or hydrogen peroxideproduction by a member of the NADPH oxidase enzyme family (Nox), andprovides uses of a composition that acts as a superoxide dismutasemimetic to decrease superoxide and/or hydrogen peroxide, wherein in oneembodiment the superoxide dismutase mimetic that decreases superoxideand/or hydrogen peroxide comprises a C60 fullerene, C₃ (tris malonicacid C60) or a malonic acid derivative, for the manufacture of apharmaceutical for: (a) ameliorating (treating, slowing the progress ofor reversing) or preventing schizophrenia, psychosis, delirium, e.g.,post-operative delirium, drug-induced psychosis, psychotic features,frailty syndrome (FS), or cognitive, learning or memory impairmentsresulting from or associated with frailty syndrome (FS), aging,depression, dementias; ameliorating or preventing traumatic warneurosis, post traumatic stress disorder (PTSD) or post-traumatic stresssyndrome (PTSS), Amyotrophic Lateral Sclerosis (ALS, or Lou Gehrig'sDisease), and/or Multiple Sclerosis (MS), and cognitive, learning ormemory impairments resulting therefrom, and frailty syndrome (FS) andaging, and the CNS inflammation of traumas and inflammation from CNSinfections; (b) ameliorating (treating, slowing the progress of orreversing) or preventing Alzheimer's disease, Lewy Body Disease,Parkinson's Disease, Huntington's Disease, Multi-infarct dementia(vascular dementia), senile dementia or Frontotemporal Dementia (Pick'sDisease), Amyotrophic Lateral Sclerosis (ALS, or Lou Gehrig's Disease),and/or Multiple Sclerosis (MS), and cognitive, learning or memoryimpairments resulting therefrom, and frailty syndrome (FS) and aging (c)increasing resistance to a CNS neurological pathology, trauma, disease,inflammation from CNS infection; and/or condition caused by and/orassociated with an increased amount of inflammation and/or oxidativestress in the CNS, or, ameliorating (treating, slowing the progress ofor reversing) or preventing a CNS inflammation caused by a CNS infectionor trauma, and cognitive, learning or memory impairments resultingtherefrom; (d) ameliorating (treating, slowing the progress of orreversing) or preventing a CNS inflammation and/or injury in aconcussive or traumatic injury and cognitive, learning or memoryimpairments resulting therefrom, and/or in an individual withpost-concussion syndrome (also known as postconcussive syndrome or PCS)and cognitive, learning or memory impairments resulting therefrom, in anindividual.

In one embodiment of the use: (a) the member of the NADPH oxidase enzymefamily (Nox) is a Nox1, Nox2, Nox3, Nox4 or Nox5 enzyme; (b) thecomposition that inhibits NFkB, IL-6, IL-6-R, a member of the NADPHoxidase enzyme family (Nox), and/or superoxide and/or hydrogen peroxideproduction by a member of the NADPH oxidase enzyme family (Nox)comprises an antisense nucleic acid, an siRNA, a miRNA or a ribozymethat binds to or hybridizes to and inhibits or decreases the activity orexpression of a nucleic acid encoding NFkB, IL-6, IL-6-R or a member ofthe NADPH oxidase enzyme family (Nox); (c) the composition that inhibitsIL-6, IL-6-R, a member of the NADPH oxidase enzyme family (Nox), and/orsuperoxide and/or hydrogen peroxide production by a member of the NADPHoxidase enzyme family (Nox) comprises an antibody the specifically bindsto NFkB, IL-6, IL-6-R or the member of the NADPH oxidase enzyme family(Nox); or (d) the infection ameliorated is a bacterial infection, aviral infection, a yeast or fungal infection, or the infectionameliorated is an HIV infection, or wherein the infection is aHaemophilus, Cryptococcus, Filobasidiella, Neisseria, Rickettsia orBorrelia infection.

The invention provides methods for ameliorating (slowing, reversing orabating) or preventing neuron or CNS or brain damage in individualshaving frailty syndrome (FS), aging, injuries, pathologies, diseases,infections and conditions causing and/or associated with an increasedamount of CNS inflammation and/or CNS oxidative stress, or acceleratingthe recovery of CNS neuron or brain damage in individuals havinginjuries, pathologies (e.g., Amyotrophic Lateral Sclerosis (ALS, or LouGehrig's Disease), and/or Multiple Sclerosis (MS)), and cognitive,learning or memory impairments resulting therefrom, diseases, infectionsand conditions causing and/or associated with an increased amount of CNSinflammation and/or CNS oxidative stress, and cognitive, learning ormemory impairments resulting therefrom, comprising:

(a) (i) providing a composition that inhibits or decreases the level ofactivation or activity of NFkB, IL-6, IL-6-R, a member of the NADPHoxidase enzyme family (Nox), and/or inhibits or decreases superoxide orhydrogen peroxide production by a member of the NADPH oxidase enzymefamily (Nox), or providing a composition that acts as a superoxidedismutase mimetic to decrease superoxide and/or hydrogen peroxide; and(ii) contacting or administering a therapeutically effective amount ofthe composition of (a) with an individual in need thereof;

(b) the method of (a), wherein the individual in need thereof is ahuman;

(c) the method of (a) or (b), wherein the composition of (a) isformulated as a pharmaceutical composition:

(d) the method of any of (a) to (c), wherein the contacting oradministering is into the CNS, or brain cortex, of the individual;

(e) the method of any of (a) to (d), wherein the individual is a human;

(f) the method of any of (a) to (e), wherein the human has Alzheimer'sdisease, Lewy Body Disease, Parkinson's Disease, Huntington's Disease,Multi-infarct dementia (vascular dementia), senile dementia orFrontotemporal Dementia (Pick's Disease), Amyotrophic Lateral Sclerosis(ALS, or Lou Gehrig's Disease), and/or Multiple Sclerosis (MS) orcognitive, learning or memory impairments resulting therefrom;

(g) the method of any of (a) to (f), wherein the composition orpharmaceutical formulation is formulated for delivery to the CNS, orbrain or a CNS neural cell, or for passing through the blood brainbarrier (BBB):

(h) the method of any of (a) to (g), wherein the composition orpharmaceutical formulation is formulated for delivery to aparvalbumin-positive GABA-ergic interneuron;

(i) the method of any of (a) to (h), wherein the composition or thepharmaceutical formulation comprises a therapeutic monoclonal antibodyspecific for and inhibitory to the activity of an NFkB, IL-6 or IL-6-Ror an NADPH oxidase (Nox) enzyme;

(j) the method of (i), wherein the therapeutic monoclonal antibody is ahumanized or a human antibody; or

(k) the method of (i) or (j), wherein the therapeutic monoclonalantibody against the IL-6-R is tocilizumab, or ACTEMRA™, or thetherapeutic monoclonal antibody is against IL-6, or is CNTO-328, ahuman-mouse chimeric monoclonal antibody (Mab) to IL-6;

(l) the method of any of (a) to (h), wherein the composition or thepharmaceutical formulation comprises a small molecule;

(m) the method of (1), wherein the small molecule comprises ano-methoxycatechol, an apocynin, a diapocynin,4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF),4-hydroxy-3′-methoxy-acetophenon, N-Vanillylnonanamide, staurosporine orrelated compounds;

(n) the method of any of (a) to (h), wherein the composition or thepharmaceutical formulation comprises an inhibitory nucleic acid moleculeto the expression of NFkB, IL-6, IL-6-R or NADPH oxidase; or

(o) the method of (n), wherein the inhibitory nucleic acid moleculecomprises an RNAi molecule, a double-stranded RNA (dsRNA) molecule, ansiRNA, a miRNA (microRNA) and/or a short hairpin RNA (shRNA) molecule,or a ribozyme, or an inhibitory fragment of an NFkB-, IL-6-, IL-6-R- orNADPH oxidase-encoding nucleic acid;

(p) the method of any of (a) to (o), wherein the member of the NADPHoxidase enzyme family (Nox) is a Nox1, Nox2, Nox3, Nox4 or Nox5 enzyme;

(q) the method of any of (a) to (p), wherein the injury is a concussiveor traumatic injury, and/or injury is post-concussion syndrome (alsoknown as postconcussive syndrome or PCS), or cognitive, learning ormemory impairments resulting therefrom; or

(r) the method of any of (a) to (p), wherein the infection is abacterial, viral or yeast infection, or the infection is an HIVinfection;

(s) the method of any of (a) to (r), wherein the composition thatinhibits or decreases the in vivo activity of interleukin-6 (IL-6) is anantibody against IL-6;

(t) the method of (s), wherein the composition that inhibits ordecreases the in vivo activity of interleukin-6 (IL-6) is IL-10;

(u) the method of (t), wherein the composition that inhibits ordecreases the in vivo activity of interleukin-6 (IL-6) is ilodecakin orTENOVIL™; or

(v) wherein superoxide dismutase mimetic that decreases superoxideand/or hydrogen peroxide comprises a C60 fullerene, C₃ (tris malonicacid C60) or a malonic acid derivative.

The invention provides methods for increasing resistance to or recoveryfrom a CNS injury, a neurological pathology, a disease, an infectionand/or a condition caused by and/or associated with an increased amountof inflammation and/or oxidative stress in the CNS or brain, orcognitive, learning or memory impairments resulting therefrom,comprising:

(a) (i) providing a composition that inhibits or decreases the level ofactivation or activity of NFkB, IL-6, IL-6-R, a member of the NADPHoxidase enzyme family (Nox), and/or inhibits or decreases superoxide orhydrogen peroxide production by a member of the NADPH oxidase enzymefamily (Nox), or providing a composition that acts as a superoxidedismutase mimetic to decrease superoxide and/or hydrogen peroxide; and(ii) contacting or administering a therapeutically effective amount ofthe composition of (a) with an individual in need thereof; and

(ii) contacting or administering a therapeutically effective amount ofthe composition of (a) with an individual in need thereof:

(b) the method of (a), wherein the individual in need thereof is ahuman;

(c) the method of (a) or (b), wherein the composition of (a) isformulated as a pharmaceutical composition;

(d) the method of any of (a) to (c), wherein the contacting oradministering is into the CNS or brain cortex of the individual;

(e) the method of any of (a) to (d), wherein the individual is a human;

(f) the method of any of (a) to (e), wherein the human has Alzheimer'sdisease, Lewy Body Disease, Parkinson's Disease, Huntington's Disease,Multi-infarct dementia (vascular dementia), senile dementia orFrontotemporal Dementia (Pick's Disease), Amyotrophic Lateral Sclerosis(ALS, or Lou Gehrig's Disease), and/or Multiple Sclerosis (MS), orcognitive, learning or memory impairments resulting therefrom;

(g) the method of any of (a) to (f), wherein the composition orpharmaceutical formulation is formulated for delivery to the CNS orbrain or a neural cell, or for passing through the blood brain barrier(BBB);

(h) the method of any of (a) to (g), wherein the composition orpharmaceutical formulation is formulated for delivery to aparvalbumin-positive GABA-ergic interneuron; (i) the method of any of(a) to (h), wherein the composition or the pharmaceutical formulationcomprises a therapeutic monoclonal antibody specific for and inhibitoryto the activity of an NFkB, IL-6 or IL-6-R or an NADPH oxidase (Nox)enzyme;

(j) the method of (i), wherein the therapeutic monoclonal antibody is ahumanized or a human antibody; or

(k) the method of (i) or (j), wherein the therapeutic monoclonalantibody against the IL-6-R is tocilizumab, or ACTEMRA™, or thetherapeutic monoclonal antibody is against IL-6, or is CNTO-328, ahuman-mouse chimeric monoclonal antibody (Mab) to IL-6;

(l) the method of any of (a) to (h), wherein the composition or thepharmaceutical formulation comprises a small molecule:

(m) the method of (l), wherein the small molecule comprises ano-methoxycatechol, an apocynin, a diapocynin,4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF),4-hydroxy-3′-methoxy-acetophenon, N-Vanillylnonanamide, staurosporine orrelated compounds;

(n) the method of any of (a) to (h), wherein the composition or thepharmaceutical formulation comprises an inhibitory nucleic acid moleculeto a nucleic acid encoding an NFkB, IL-6, IL-6-R or NADPH oxidase; or

(o) the method of (n), wherein the inhibitory nucleic acid moleculecomprises an RNAi molecule, a double-stranded RNA (dsRNA) molecule, ansiRNA, an miRNA (microRNA) and/or a short hairpin RNA (shRNA) molecule,or a ribozyme, or an inhibitory fragment of an NFkB-, IL-6-, IL-6-R- orNADPH oxidase-encoding nucleic acid sequence;

(p) the method of any of (a) to (o), wherein the member of the NADPHoxidase enzyme family (Nox) is a Nox1, Nox2, Nox3, Nox4 or Nox5 enzyme;or

(q) the method of any of (a) to (p), wherein the method increasesresistance to a concussive or traumatic injury, and/or the methodincreases resistance to or ameliorate the effects of post-concussionsyndrome (also known as postconcussive syndrome or PCS), or cognitive,learning or memory impairments resulting therefrom;

(r) the method of any of (a) to (q), wherein the composition thatinhibits or decreases the in vivo activity of interleukin-6 (IL-6) is anantibody against IL-6;

(s) the method of (r), wherein the composition that inhibits ordecreases the in vivo activity of interleukin-6 (IL-6) is IL-10;

(t) the method of (s), wherein the composition that inhibits ordecreases the in vivo activity of interleukin-6 (IL-6) is ilodecakin orTENOVIL™; or

(u) the method of(s), wherein the superoxide dismutase mimetic thatdecreases superoxide and/or hydrogen peroxide comprises a C60 fullerene,C₃ (tris malonic acid C60) or a malonic acid derivative.

The invention provides methods for purifying C60 fullerene derivatives,including C₃ (tris malonic acid C60) and other malonic acid derivativescomprising

(i) (a) dissolving an impure powder form of C₃ (tris malonic acid C60fullerene) or other malonic acid derivatives in dilute sodium hydroxide(NaOH) solution at a concentration of between about 1 mM to 400 mM atabout 4 degrees C. with stirring;

(b) adding a second solution of NaOH more concentrated than the diluteNaOH solution in step (a) drop-wise to the solution of step (a) toachieve an approximately neutral pH;

(c) incubating the solution of step (b) at 4 degrees C. in the dark forapproximately 0.5 to 3 hours;

(d) centrifuging the solution after the incubating of step (c) toproduce a clear dark red supernatant and a solid light pink pellet;

(e) removing the supernatant to a different container;

(f) incubating the supernatant removed in step (e) at 4 degrees C. foran additional about 3 to 4 hours; and

(g) (1) re-centrifuging to remove substantially all or all undissolvedmaterial to generate a pellet and a solution comprising purified C₃,wherein the pellet comprises an insoluble waxy material containingcontaminant and small amounts of residual C₃, or (2) filtering thesample through a filter which allows only aqueous solutions to pass,thereby removing an insoluble waxy contaminant after solubilization indilute NaOH, thereby generating a solution comprising purified C₃; or

(ii) the method of (i), wherein the purified C₃ solution is furthertreated to remove a minor amount of volatile contaminant by vacuumdistillation or by bubbling an inert gas through the solution.

The invention provides methods for purifying a C60 fullerene derivative,C₃ (tris malonic acid C60) or other malonic acid derivatives comprising

(a) providing a solution comprising an impure powder form of C₃ (trismalonic acid C60) or other malonic acid derivative; and

(b) providing an antibody directed against the C60 fullerene derivative,C₃ (tris malonic acid C60) or other malonic acid derivative; and

(c) isolating the C60 fullerene derivative. C₃ (tris malonic acid C60)or other malonic acid derivative by incubating the antibody with the C60fullerene derivative, C₃ (tris malonic acid C60) or other malonic acidderivative under conditions wherein the antibody specifically binds tothe C60 fullerene, to C₃ (tris malonic acid C60) or to another malonicacid derivative; or

(ii) the method of (i), wherein an antibody-C60 fullerene, antibody-C₃(tris malonic acid C60) or antibody-malonic acid derivative complex ispurified by gel electrophoresis purification, HPLC, immunoprecipitation,column chromatography, differential centrifugation or affinity columnchromatography.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

All publications, patents, patent applications cited herein are herebyexpressly incorporated by reference for all purposes.

DESCRIPTION OF DRAWINGS

FIGS. 1A-1E illustrate experimental results showing that ketamineexposure in primary neuronal cultures increases superoxide and/orhydrogen peroxide production and induces the loss of parvalbuminimmunoreactivity, as described in detail in Example 1, below: FIG. 1A,FIG. 1B, and FIG. 1C: Confocal images of representative fields depictinga parvalbumin-positive (PV)-interneuron and surrounding neurons treatedin the absence of ketamine (control) (FIG. 1A), the presence of ketamine(FIG. 1B), and co-exposure to ketamine and muscimol (FIG. 1C); FIG. 1Dand FIG. 1E: graphic illustration of quantification results for DHE(FIG. 1D), and PV (FIG. 1E) fluorescence; as discussed in detail inExample 1, below.

FIGS. 2A-2B graphically illustrates experimental results showing thatremoval of superoxide or inhibition of NADPH oxidase (Nox) activationprevents superoxide increase and reduction of parvalbumin and glutamatedecarboxylase 67 (GAD67) in PV-interneurons in culture: cultures weretreated with ketamine as in FIGS. 1A-1E in the absence or presence ofthe carboxyfullerene-based SOD-mimetic C₃ (20 μM) or the Nox inhibitorapocynin (0.5 mM), and quantification results for oxidized DHEfluorescence (FIG. 2A), and for parvalbumin and GAD67 fluorescence inPV-interneurons (FIG. 2B) graphically illustrated; as discussed indetail in Example 1, below.

FIGS. 3A-3B illustrate in graphics and images experimental resultsshowing that in vivo ketamine treatment increases Nox and p22^(phox)protein expression in brain membranes, and increases the levels ofapocynin-inhibitable Nox activity in synaptosomes: FIG. 3A illustratesboth membrane fractions as analyzed for the expression of the indicatedproteins (Nox2, Nox4, p22^(phox), and Actin) by image of Western blots(insert to FIG. 3A), and FIG. 3A bar graph graphically represent thequantification of Western blot data normalized for actin content; FIG.3B bar graph illustrates data showing increased Nox activity wasobserved in synaptosomal preparations from ketamine treated animals; asdiscussed in detail in Example 1, below.

FIGS. 4A-4G illustrates in graphics and images experimental resultsshowing that pretreatment of animals with the Nox inhibitor apocynin, orwith the SOD-mimetic (C₃), reduces superoxide and/or hydrogen peroxideproduction and prevents the loss of parvalbumin immunoreactivity inducedby ketamine in mouse prefrontal cortex, as described in detail inExample 1, below; animals were treated with ketamine as in FIGS. 3A-3B,and coronal sections comprising the prelimbic and infralimbic regionswere analyzed: FIG. 4A: confocal images showing parvalbumin and GAD67expression in PV-interneurons (upper panels are saline controls, withlower panels the ketamine treated samples); graph bar of FIG. 4Crepresents the quantification of parvalbumin and GAD67 meanfluorescence/cell for the region normalized by the means of salinetreated animals; FIG. 4B and FIG. 4D: animals were treated with apocyninin the drinking water for 1 week, or during one month with theSOD-mimetic C₃ delivered by mini-pumps before ketamine treatment; asdiscussed in detail in Example 1, below. FIGS. 4F and 4G illustrate inimages the effects of ketamine on oxidized DHE and parvalbuminexpression in other brain regions such as the hippocampal CA3 region(FIG. 4F) and the reticular nucleus of the thalamus (FIG. 4G), asdiscussed in detail in Example 1, below.

FIGS. 5A-5B illustrate in graphics and images experimental resultsshowing increasing GABA_((A))-mediated inhibition prevents the decreasein GAD67 expression in parvalbumin-positive (PV)-interneurons afterketamine treatment in primary neuronal cultures: cultures were treatedwith ketamine in the absence or presence of muscimol as in FIGS. 1A-1E,above, and GAD67 immunofluorescence in PV-interneurons was analyzed(FIG. 5A, left panel control; middle panel ketamine treatment; rightpanel ketamine and muscimol treatment); FIG. 5B: graph bar representsthe quantification of mean fluorescence/cell as a percent of control; asdiscussed in detail in Example 1, below.

FIGS. 6A-6B illustrate in graphics and images experimental resultsshowing that ketamine treatment increases Nox2 expression in primaryneuronal cultures: FIG. 6A illustrates confocal images showing theincrease in Nox2 immunoreactivity after 24 h of treatment with ketaminein primary cultured neurons (upper three panels control, lower threepanels ketamine treated) with MAP-2 immunoreactivity used as a markerfor neurons; FIG. 6B: inset shows image of Western blots prepared formcultures treated as in FIG. 6A, showing increase in Nox2 protein level,with bar graph schematically illustrating-summarizing the data from thisstudy; as discussed in detail in Example 1, below.

FIGS. 7A-7B graphically illustrate experimental results showing ketamineeffects on synaptosomal O₂ consumption by Nox(s) and mitochondria: FIG.7A: graphically summarizes data showing oxygen consumption bysynaptosomal Nox(s) from cortex of saline or ketamine injected mice wasinduced by the addition of 5 mM NADPH to samples containing synaptosomalprotein; the inset in FIG. 7A graphically illustrates data showing theapocynin dependent inhibition of Nox activity; FIG. 7B graphicallysummarizes data showing ketamine treatment did not affect synaptosomalmitochondria; respiratory function of synaptosomal mitochondria in thesame preparations was then evaluated by the subsequent addition ofNAD⁺-linked substrates followed by the addition of the F₀F₁-ATPaseinhibitor oligomycin to attain State 4 respiration, and the maximalmitochondria respiration was initiated by the addition of theprotonophore uncoupling agent, CCCP, as illustrated in FIG. 7B; asdiscussed in detail in Example 1, below.

FIG. 8 illustrates in graphics and images experimental results showingketamine-mediated decrease in parvalbumin and GAD67 immunoreactivity inPV-interneurons of the PFC is prevented by apocynin treatment; left ninepanels are confocal images of parvalbumin and GAD67 stained sections ofthe prefrontal region depicting the decrease in immunoreactivity inducedby the two-day ketamine treatment; right bar graph representsmeans+/−SEM values expressed as % of control (saline) conditions; asdiscussed in detail in Example 1, below.

FIG. 9 left and right panels graphically illustrate data demonstratingthat ketamine exposure induced a pronounced increase in DHE oxidationboth in vivo (in the prefrontal cortex, PFC) and in cultures, asdescribed in detail in Example 1, below; see also explanation for FIGS.1A-1E and 4A-4G.

FIG. 10 all four panels illustrate representative confocal images fromthe experiments illustrated in FIG. 9, as described in detail in Example2, below.

FIG. 11 graphically illustrates data from primary cultures exposed toketamine in the presence of the pan-GABA_((A)) agonist muscimol (10 μM),as described in detail in Example 1, below; see also explanation forFIGS. 5A-5B.

FIG. 12 graphically illustrates data demonstrating that a SOD mimetic(C₃), or the Nox inhibitor apocynin (Apo) prevented ketamine-mediatedsuperoxide and/or hydrogen peroxide production in cultures, as describedin detail in Example 1, below; see also explanation for FIG. 2A-2B.

FIGS. 13A-13B in images and graphics illustrate data showing that Nox2is expressed in cortex and ketamine treatment increased its expressionin vitro and in vivo; ketamine treatment increased the expression ofNox2 in cultures, as shown in the confocal images of the six panels ofFIG. 13A; and also increased Nox2 and p22^(phox) in cortical particulatefractions from ketamine treated animals, as graphically shown in FIG.13B; the inset illustrating Western blots of levels of the indicatedproteins in the various samples, as described in detail in Example 1,below; see also explanation for FIGS. 3A-3B and 6A-6B.

FIG. 14A illustrates schematically an exemplary experimental schemeusing synaptosomes, and FIG. 14B, which graphically summarizes datashowing dose-dependent inhibition of NADPH-stimulated O₂ consumption byapocynin; inset of FIG. 14B graphically illustrates inhibition of Nox byapocynin; as described in detail in Example 1, below; see alsoexplanation for FIGS. 7A-7B.

FIG. 15 schematically illustrates data showing that SOD mimetic andapocynin prevented ketamine effects on PV-interneurons in culture; asdescribed in detail in Example 1, below; see also explanation for FIGS.2A-2B.

FIGS. 16(i)-(iii) illustrate data showing that synaptosomal Nox is anactive source of free radicals: EPR spectra recorded after 1 hrincubation of approximately (˜) 10 mg synaptosomal protein isolated frommouse brain at 37° C. in the absence of Nox or mitochondria substratesis shown in FIG. 16(i), in the presence of 10 mM malate+10 mM pyruvateis shown in FIG. 16 (ii), or 200 mM digitonin+5 mM NADPH is shown inFIG. 16(iii); as described in detail in Example 1, below; see alsoexplanation for FIGS. 7A-7B.

FIG. 17 illustrates data schematically and by image showing involvementof Nox activation in ketamine effects on PV-interneurons in vivo, wheremice were treated with ketamine in the absence or presence of eitherapocynin or the brain-permeable SOD mimetic (C₃), and ketamine reducedparvalbumin and GAD67 expression in the PFC, as illustrated in FIG. 17,left two confocal images and graphic data summary; and treatment with C₃or apocynin prevented the loss of parvalbumin in PV-interneurons andreduced DHE oxidation, as illustrated in FIG. 17, right six confocalimages and two graphic data summaries; as described in detail in Example1, below; see also explanation for FIGS. 4A-4G.

FIG. 18 illustrates data showing that IL-6, when applied for 24 h,increased the levels of Nox expression and DHE oxidation (top panels)and decreased the immunoreactivity of GAD67 and parvalbumin (bottompanels), and these effects were prevented by the Nox inhibitor apocynin,as described in detail in Example 2, below.

FIG. 19 schematically illustrates data showing the results of treatingcultured neurons with a subthreshold concentration of ketamine in theabsence or presence of IL-6, as described in detail in Example 2, below.

FIG. 20 schematically illustrates data showing that muscimol preventsonly ketamine-mediated induction of Nox2 in primary cultures; cultureswere treated with ketamine or IL-6 in the absence or presence ofmuscimol, as described in detail in Example 2, below.

FIG. 21 schematically illustrates data showing that only IL-6 mRNAexpression, but not IL-1β or TNFα, was induced by ketamine in cultures;cultures were treated with ketamine for varying periods of time and mRNAwas extracted, as described in detail in Example 2, below.

FIG. 22 schematically illustrates data showing a significant inductionof Nox activity in by IL-6 in synaptosomal preparations, as described indetail in Example 2, below.

FIG. 23 graphically illustrates data showing the slow reversal ofketamine effects on PV-interneurons in vivo, as described in detail inExample 2, below.

FIGS. 24A-24B graphically illustrate data showing an analysis of theprelimbic region that showed that deletion of Nox2 prevented theincrease in superoxide induced by ketamine, as shown by the datagraphically illustrated in FIG. 24A (top graphic), and protected thephenotype of PV-interneurons, as shown by the data graphicallyillustrated in FIG. 24B (lower graphic), as described in detail inExample 2, below.

FIG. 25 graphically illustrates data from neuronal cultures exposed toketamine and IL-6, which shows that blocking activity of thetranscription factor NFκB using SN50 blocks induction and activation ofNox2, as assessed by DHE oxidation, as described in detail in Example 2,below.

FIG. 26 graphically illustrates data testing whether glial cells wereresponsible for the increase in IL-6 upon ketamine exposure; the NMDA-Rantagonist ketamine was applied to neurons in the absence of theastrocytic layer, and the PV-interneuronal population analyzed; ketamineproduced a similar increase in DHE oxidation and loss of phenotype ofPV-interneurons in the presence or absence of the astrocytic layer, asdescribed in detail in Example 2, below.

FIGS. 27A-27B by graphs and imaging illustrate data where primaryneuronal cultures were exposed to ketamine in the absence of theastrocytic monolayer and in the presence of an anti-mouse IL-6 blockingantibody produced in goat (anti-mIL-6); FIG. 27A, four panelsillustrating confocal images of cells showing that increasingconcentrations of anti-mIL-6 prevented the decrease in parvalbumin (PV)and GAD67 after 24 h of ketamine exposure. Bottom bar graph shows thefluorescence quantification of both antigens in PV-interneuronsexpressed as % of control; FIG. 27B: four panels illustrating confocalimages showing that increasing concentrations of anti-mIL-6 preventedthe increase in oxidized DHE cause by ketamine exposure. Bottom bargraph shows results for fluorescence quantification of oxidized DHEexpressed as % of control, as described in detail in Example 2, below.

FIG. 28 by graphs and imaging illustrates data where primary neuronalcultures were exposed to ketamine in the absence of the astrocyticmonolayer and in the presence of an anti-mouse IL-6 blocking antibodyproduced in rat (anti-mIL-6); FIG. 28 upper six panels illustrateconfocal images of cells showing that increasing concentrations ofanti-mIL-6 prevented the decrease in parvalbumin (PV) and GAD67 after 24h of ketamine exposure; and the bar graph shows results for fluorescencequantification of both antigens in PV-interneurons expressed as % ofcontrol, as described in detail in Example 2, below.

FIG. 29A illustrates results showing that ketamine exposure in vivo ontwo consecutive days leads to increased mRNA expression of IL-6 inbrain, without affecting the expression levels of IL-1β or TNFα; FIG.29B, C by graphs and imaging illustrates data showing that ketamine doesnot lead to increased DHE oxidation and loss of GABAergic phenotype ofPV-interneurons in IL-6−/− mice, as described in detail in Example 2,below.

FIGS. 30A-30B graphically illustrate data showing that ketamine-inducedIL-6 release directly activates Nox; FIG. 30A: EPR assessment ofsuperoxide production in live cultures upon treatment with ketamine;primary cultures were exposed to ketamine for the times indicated in theabsence or presence of an anti-mouse IL-6 blocking antibody produced inrat, at the indicated times, the coverslips were transferred to a quartzchamber and superoxide production was followed by EPR spectroscopy usingthe spin-trap DIPPMPO; FIG. 30B: IL-6 increased basal NADPH oxidaseactivity in forebrain synaptosomes isolated from 3 month-old mouseforebrains accumulation of superoxide during the first 6 min wasanalyzed using the spin trap DEPMPO, as described in detail in Example2, below.

FIGS. 31A-31C illustrate data analyzing the presence and expression ofisoforms of Nox and tested whether Nox activity contributes tosuperoxide levels in the aged brain: FIG. 31A illustrates four panels ofgels of mRNAs for Nox1, Nox2, Nox3, Nox4, Nox5 and p22^(phox), asindicated, showing levels were increased in several brain regions ofaged mice; FIG. 31B: Western blot analysis of young and old forebrainproteins demonstrated an increase in Nox2, Nox4 and p22 protein content,with this data also graphically illustrated; FIG. 31C: Western blotanalysis showing the specificity of the antibodies used for Nox2 wasconfirmed in wild type and gp91phox−/− forebrain extracts, as describedin detail in Example 3, below.

FIG. 32A illustrates nine panels of confocal images of cells showingthat aging (old) mice showed increased immunostaining for Nox proteins;immunohistochemistry performed on brain slices from young and oldanimals revealed increased Nox2; Nox2 expression was increased inneurons and astrocytes in old animals; confocal imaging of the neuronalmarker, MAP2 (red), astrocytes marker, GFAP (red), gp91^(phox) (green)and merged images; antibodies were polyclonal anti-MAP2, polyclonalanti-GFAP, and monoclonal 54.1 gp91^(phox); FIG. 32B illustrates ninepanels of confocal images of cells showing that aging (old) mice showedincreased immunostaining for Nox proteins; immunohistochemistryperformed on brain slices from young and old animals revealed increasedNox4; Nox4 expression was increased in neurons and astrocytes in oldanimals; confocal imaging of the neuronal marker, MAP2 (red), GFAP(red), Nox4 (green) and merged images, antibodies were polyclonalanti-MAP2, polyclonal anti-GFAP, and monoclonal anti-Nox4 antibody, sdescribed in detail in Example 3, below.

FIGS. 33A-33D illustrate four confocal images and graphics showing dataof in vivo elevated levels of superoxide production in the pyramidallayer of CA1 in the aged hippocampus, which were prevented by oraladministration of the brain-permeable SOD mimetic C₃, and by the Noxinhibitor apocynin, which is summarized in the graphic below theconfocal images; and including graphic of Nox activity by oximetry withan inset graph showing the relationship of Nox activity and apocyninconcentration, and an EPR of superoxide production by Nox; FIG. 33Cillustrates Nox specific activity on young and old animals; FIG. 33Dillustrate mitochondrial specific activity; as described in detail inExample 3, below.

FIGS. 34A-34B illustrate confocal images of cortical neurons afterexposure to IL-6; FIG. 34A illustrates six confocal image panels showingthat the phosphorylation of the protein kinase Jak2 increased; FIG. 34Billustrates nine confocal image panels showing that prolonged exposureto the interleukin increased production of superoxide and increased theexpression of Nox2 in neurons; the role of Nox2 activation in theincrease in DHE oxidation was confirmed by co-exposure to the Noxinhibitor apocynin (FIG. 34B bottom panels), as described in detail inExample 3, below.

FIGS. 35A-35C illustrates an image and graphics showing data that IL-6treatment in vivo increases Nox2 mRNA in brain as well as Nox proteinand activity in synaptosomes: illustrates a gel RNA image of Nox2 mRNAdetected by RT-PCR from four month old mice treated with either salineor with IL-6, and brains were either processed for RNA or forsynaptosomal preparation; FIG. 35 illustrates immunoblots of Nox2 andp22 (and control actin) synaptosomal proteins in samples with andwithout IL-6 treatment separated on 10% SDS-PAGE gels, and graphicallysummarizes the data from the immunoblots; FIGS. 35A-35C graphicallyillustrate Nox activity in synaptosomes with or without apocynin, asindicated; as described in detail in Example 3, below.

FIG. 36 illustrates the absorption spectra of pure C₃ prepared by theBingel procedure, as described in detail in Example 2, below.

FIG. 37 illustrates absorption spectra of Regis C₃ prior to clean-up, asdescribed in detail in Example 2, below.

FIG. 38A and FIG. 38B illustrates absorption spectrum of C₃ (Regis)after purification using the exemplary protocol (method) of thisinvention at 2 dilutions to allow all wavelengths of the spectrum to beviewed on scale, as described in detail in Example 2, below.

FIG. 39A and FIG. 39B illustrate data demonstrating neuroprotectionagainst NMDA toxicity by a lot of pure C₃ using the exemplarypurification protocol of this invention, as described in detail inExample 2, below.

FIGS. 40A-40C illustrate data demonstrating age-related reduction innumber of parvalbumin-interneurons in the prefrontal cortex: fluorescentstaining for markers is shown in FIG. 40A; coronal sections comprisingthe regions between Bregma 2.0 and 1.3 are as shown in FIG. 40B, and thecumulative results for the expression of each CBP are shown in FIG. 40C,as described in detail in Example 4, below.

FIGS. 41A-41C illustrate data demonstrating age-related decrease ofPV-interneurons in prefrontal and hippocampal regions: long-term chronictreatment with an SOD-mimetic prevents interneuron loss: coronal brainslices of young (YM) and old (OM) male mice were stained for parvalbuminand total PV-positive cell counts were evaluated across 4 slices of theprelimbic region (PFC) and hippocampal regions CA1, CA3 and dentategyrus (DG), as shown in FIG. 41A; aging was accompanied by astatistically significant decrease in PV-interneuron number in allregions analyzed, as shown in FIG. 41B; treatment of animals from middleage with the SOD-mimetic C3 (OM+C3) prevented the reduction ofPV-interneuron numbers in CA1 and CA3, but not in DG as shown in FIG.41C, as described in detail in Example 4, below.

FIGS. 42A-42C illustrate data demonstrating that the aged prefrontalcortex is more vulnerable to the effects of ketamine on parvalbumin andcalbindin interneurons: brain coronal sections from animals treated withsaline or ketamine were double stained for each CBP and GAD67; FIG. 42A:effect of ketamine on the average mean intensity per cell for each CBPin the PFC region; FIG. 42B: analysis of the mean intensity per cell forGAD67 content analyzed in each CBP stained cell; FIG. 42C: confocalimages obtained with a 40× objective depicting the effects of ketamineon the immuno-fluorescence for PV and GAD67 in the PFC region of youngand old animals, as described in detail in Example 4, below.

FIGS. 43A-43-B illustrate data demonstrating that aging increases thevulnerability to Nox-dependent loss of phenotype of PV-interneurons andsensitivity to low doses of an anesthetic ketamine: FIG. 43A data showsthere is enhanced vulnerability of the remaining neurons toloss-of-phenotype (and loss of inhibitory function) in old mice (ascompared to young mice) in response to even sub-anesthetic doses of ananesthetic; aging also increases the sensitivity of old mice (ascompared to young mice) to ketamine at 20, 30 and 40 mg/kg, as shown inFIG. 43B, as described in detail in Example 4, below.

FIG. 44 illustrates data demonstrating that plasma IL-6 is increasedwith aging or after intraperitoneal (i.p.) administration of IL-6, IL-6was assayed by ELISA, mice were then given a direct intraperitoneal (IP)injection IL-6 on two consecutive days, and plasma IL-6 was assayed 16hours (hr) after the last injection, as described in detail in Example4, below.

FIG. 45 illustrates data demonstrating that NFkB (p65) activity asmeasured in brain nuclear extracts from old wild-type (WT) (“CTL”, orcontrol) versus old IL-6−/− mice (“IL-6 KO”, or IL-6 knockout) by anELISA kit for the p65 subunit of NFkB, with “no oligo” and “mutantoligo” controls, as described in detail in Example 4, below.

FIG. 46 illustrates data demonstrating that RNA expression of IL-1β andTNFα was measured in brain extracts from old wild-type and old IL-6−/−mice, indicating that lack of IL-6 expression in the IL-6−/− mice doesnot modify expression of IL-1β or TNFα, as described in detail inExample 4, below.

FIG. 47 illustrates data demonstrating that Nox-dependent superoxideproduction is lower in synaptosomes from IL-6-KO old mice compared toage-matched wild-type controls, as measured by EPR, with spectraillustrated in FIG. 47, left, as graphically illustrated in FIG. 47,right, as described in detail in Example 4, below.

FIG. 48 illustrates data demonstrating that performance of IL-6deficient old mice compared to age-matched (old) wild-type controls on arotorod test showed that in day 2 and day 3 test samples the presence ofIL-6 decreased the level of performance, as described in detail inExample 4, below.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

The invention provides compositions and methods for the amelioration orprevention, including the treatment of, a CNS inflammation, psychosis,delirium, schizophrenia, depression and/or dementias, traumatic warneurosis, post traumatic stress disorder (PTSD) or post-traumatic stresssyndrome (PTSS), and cognitive, learning or memory impairments resultingtherefrom, in an individual, e.g., in humans. The invention providescompositions and methods for the amelioration or prevention of diseasesor conditions caused by diminished activity of parvalbumin-positiveGABA-ergic interneurons in the cortex and which are caused by activationof signaling mechanisms that induce and activate any member of the NADPHoxidase family (Nox). The invention provides compositions and methods toinhibit or decrease (amount or rate of) activation of any member of theNADPH oxidase family (Nox) family, and/or block or inhibit NFkB and/orinterleukin-6 (IL-6)-mediated NADPH oxidase (Nox) activation andinduction, thus ameliorating or preventing or treating a CNSinflammation, psychosis, delirium, schizophrenia, depression and/ordementias, traumatic war neurosis, post traumatic stress disorder (PTSD)or post-traumatic stress syndrome (PTSS), Amyotrophic Lateral Sclerosis(ALS, or Lou Gehrig's Disease), and/or Multiple Sclerosis (MS), andcognitive, learning or memory impairments resulting therefrom, andfrailty syndrome (FS) and aging. In one embodiment, blocking orinhibiting NFkB or interleukin-6 (IL-6)-mediated NADPH oxidase (Nox)activation and induction comprises blocking or inhibitinginterleukin-6-R (IL-6-R) activation by IL-6, which can comprise blockingor inhibiting interleukin-6-R (IL-6-R) binding with an IL-6-R activationligand, e.g., the IL-6-R ligand IL-6. In one embodiment, the inventionprovides for administering a superoxide dismutase (SOD) mimetic such asa malonic acid derivative, e.g., the fullerene C60, or thecarboxyfullerene-based SOD-mimetic C₃ to decrease superoxide levels in acell of the CNS.

The inventors have discovered and demonstrated that specificinflammatory pathways are involved in alterations in the CNS, e.g., thebrain, that are known to be associated with a CNS inflammation,psychosis, delirium, schizophrenia, depression and/or dementias,traumatic war neurosis, post traumatic stress disorder (PTSD) orpost-traumatic stress syndrome (PTSS), Amyotrophic Lateral Sclerosis(ALS, or Lou Gehrig's Disease), and Multiple Sclerosis (MS), andcognitive, learning or memory impairments resulting therefrom, inhumans. These alterations include dysfunction of a critical set ofneurons—the parvalbumin-positive GABA-ergic interneurons—in the cortexof the brain. Using a well-established mouse model ofschizophrenia/psychosis the inventors specifically demonstrated thatNADPH oxidase, an inflammatory enzyme complex, is induced and activatedin neurons in brain in this mouse model. The inventors then demonstratedthat NADPH oxidase (Nox) is responsible for dysfunction of theparvalbumin-positive interneurons, and that inhibiting NADPH oxidaserescues these same neurons. The inventors also show that eliminatingsuperoxide/hydrogen peroxide produced by NADPH oxidase or other sourcesrescues these same neurons; thus, in one embodiment the inventionprovides compositions and methods for rescuing parvalbumin-positiveinterneurons by e.g., inhibiting or decreasing the activity of anymember of the NADPH oxidase either directly or indirectly, e.g., bydirectly or indirectly inhibiting or decreasing the activity of IL-6and/or IL-6-R. The inventors demonstrated that interleukin-6 (IL-6) isresponsible for the induction and activation of NADPH oxidase in thismodel. Finally, the inventors demonstrated that administration ofcomposition that acts as a superoxide dismutase mimetic to decreasesuperoxide and/or hydrogen peroxide has a cytoprotective effect in theCNS. See e.g. Examples 1 through 3, below.

Thus, the invention provides compositions and methods to decrease NFkB,IL-6 and/or Nox enzyme levels and/or activity, or to decrease superoxideand/or hydrogen peroxide levels in the CNS, to treat patients withpsychosis, schizophrenia, and many dementing disorders, CNSinflammation, delirium, depression, traumatic war neurosis, posttraumatic stress disorder (PTSD) or post-traumatic stress syndrome(PTSS), Amyotrophic Lateral Sclerosis (ALS, or Lou Gehrig's Disease),and/or Multiple Sclerosis (MS), and cognitive, learning or memoryimpairments resulting therefrom. The invention provides compositions andmethods using a therapeutic monoclonal antibody against IL-6 receptors,e.g., tocilizumab (ACTEMRA™), or the therapeutic monoclonal antibody isagainst IL-6, e.g., is CNTO-328, a human-mouse chimeric monoclonalantibody (Mab) to IL-6 (Centocor, Inc., Horsham, Pa.). Thus, theinvention provides compositions and methods to inhibit any member of theNADPH oxidase enzyme family and/or IL-6 because they are therapeutictargets in psychosis, schizophrenia, dementias, CNS inflammation,delirium, depression, traumatic war neurosis, post traumatic stressdisorder (PTSD) or post-traumatic stress syndrome (PTSS), AmyotrophicLateral Sclerosis (ALS, or Lou Gehrig's Disease), and/or MultipleSclerosis (MS), and cognitive, learning or memory impairments resultingtherefrom.

An additional embodiment comprises compositions and methods fordecreasing NFkB or IL-6 levels by using anti-NFkB or anti-IL-6antibodies, respectively (which can be monoclonal, recombinant,fragments, humanized, and the like), such as CNTO-328, a human-mousechimeric monoclonal antibody (Mab) to IL-6 (Centocor, Inc., Horsham,Pa.), which recognizes human IL-6 and enhances its degradation. CNTO-328is reported to have a plasma half-life of roughly 17 days and thus inone embodiment is administered at periods from about 2 to 6 weeks,depending on individual variation in metabolism of the antibody.

An additional embodiment comprises compositions and methods for loweringIL-6 levels or effects is through administration of IL-10. IL-10regulates production and thus levels of LI-6. In one aspect, theinvention provides for direct administration of IL-10, for example as ahumanized IL-10 preparation (e.g., ilodecakin, TENOVIL™,Schering-Plough, Kenilworth, N.J.) to lower IL-6 production. Anadditional embodiment comprises use of small-molecule IL-10 mimetics (asIL-10 agonists—mimics) to lower IL-6 levels or effects.

This invention for the first time identifies novel pathways, includingIL-6 to any member of the NADPH oxidase enzyme family, to superoxideand/or hydrogen peroxide production, which leads to dysfunction of theinhibitory neurons associated with these vulnerable circuits, e.g.,involved in psychosis, schizophrenia, and many dementing disorders, CNSinflammation, delirium, depression, traumatic war neurosis, posttraumatic stress disorder (PTSD) or post-traumatic stress syndrome(PTSS), Amyotrophic Lateral Sclerosis (ALS, or Lou Gehrig's Disease),and/or Multiple Sclerosis (MS), and cognitive, learning or memoryimpairments resulting therefrom, and frailty syndrome (FS) and aging.This invention provides alternative embodiments using novel therapeutictargets to treat, ameliorate or prevent pathologies or inflammation inthe central nervous system (CNS), or the brain, e.g., schizophrenia,psychosis, delirium, e.g., post-operative delirium, drug-inducedpsychosis, psychotic features associated with frailty syndrome (FS),aging; depression and/or dementias, traumatic war neurosis, posttraumatic stress disorder (PTSD) or post-traumatic stress syndrome(PTSS), Amyotrophic Lateral Sclerosis (ALS, or Lou Gehrig's Disease),and/or Multiple Sclerosis (MS), and cognitive, learning or memoryimpairments resulting therefrom, and frailty syndrome (FS) and aging;wherein the novel therapeutic target include for example: NFkB, IL-6,IL-6-R or any member of the NADPH oxidase, to decrease superoxide and/orhydrogen peroxide production by any member of the NADPH oxidase. Inalternative embodiments, the invention for the first time providesmechanistic treatments (as opposed to symptomatic treatments), includingcompositions and methods, for these important neuropathologicalconditions.

The inventors have verified these findings on an art-acceptedexperimental animal model for schizophrenia and psychosis. This model iscommonly used to study schizophrenia and psychosis, and reproduces amajority of the positive and negative symptoms associated with theseconditions, and also recapitulates much of the neuroanatomical changesfound in individuals with schizophrenia, psychosis and in individualsshowing greater vulnerability to drug-induced or post-operativepsychotic episodes. This invention provides compositions and methods toameliorate frailty syndrome (FS), aging, schizophrenia, situationalpsychosis (post-operative, drug-induced, depression-associated, indementia) and dementia associated with neurodegenerative diseases suchas Alzheimer's disease, Lewy Body Disease. Parkinson's Disease,Huntington's Disease, Multi-infarct dementia, senile dementia orFrontotemporal Dementia, Amyotrophic Lateral Sclerosis (ALS, or LouGehrig's Disease), and/or Multiple Sclerosis (MS), and cognitive,learning or memory impairments resulting therefrom, and/orneurodegeneration associated with infection or trauma, e.g., humanimmunodeficiency virus (HIV) infection, or bacterial, yeast and/or viralinfections from, e.g., Haemophilus, Cryptococcus, Filobasidiella,Neisseria, Rickettsia or Borrelia infections.

This invention provides treatments (e.g., formulations and methods)using any monoclonal antibody against IL-6 receptor, e.g.,tociluzimab—which is already clinically approved.

This invention demonstrates that selective dysfunction of theparvalbumin-immunoreactive subpopulation of fast-spiking, inhibitoryinterneurons in cortex is an underlying cause for both the psychoticepisodes observed in schizophrenic patients. While the invention is notdependent on any particular mechanism of action, the invention is basedin part on the observation that decreased expression of GAD67 andparvalbumin (PV) in these PV-interneurons is a consistent finding inpostmortem brain from schizophrenic patients, and that schizophrenicpatients exhibit neurocognitive evidence of dysfunctional GABAinhibitory systems. Sub-anesthetic doses of NMDA receptor (NMDA-R)antagonists (e.g. ketamine) were used to model its neurocognitivefeatures because they reproduce both negative and positive symptoms ofschizophrenia.

Intraperitoneal (ip) injection of sub-anesthetic ketamine on twoconsecutive days in mice caused a significant induction of NADPH oxidase(Nox; or Nox2 the respiratory burst oxidase), in brain, and thisinduction was accompanied by a significant increase in Nox-dependentsuperoxide and/or hydrogen peroxide production in neurons in vivo and invitro, and in synaptosomes.

In addition, treatment of mice with a brain-permeable superoxide (SOD)mimetic or the selective Nox inhibitor, apocynin, not only blockedketamine-induced superoxide and/or hydrogen peroxide production, butfully rescued the phenotype changes in PV-interneurons, thusdemonstrating in vivo the efficacy of embodiments of the compositionsand methods of the invention.

It was also determined that administration of interleukin-6 (IL-6)reproduced the ketamine effects in vivo and in vitro; IL-6 actsdownstream of ketamine, linking known CNS inflammatory changes inschizophrenia with altered inhibitory neurotransmitter systems. Theinvention demonstrates that ketamine results in induction of Nox2 inneurons through IL-6 signaling, and that Nox2-dependent neuronalsuperoxide and/or hydrogen peroxide production mediates the loss ofphenotype (i.e. decreased GAD67 and parvalbumin expression) and function(altered electrophysiology) of PV-positive interneurons in prefrontalcortex.

Ketamine treatment in animals or neuronal cultures increases expressionof Nox2 and Nox-dependent superoxide and/or hydrogen peroxideproduction, and leads to loss of the GABAergic phenotype ofPV-interneurons. Prevention of ketamine-induced disinhibition using theGABA_((A)) agonist muscimol attenuated these effects in primarycultures, whereas IL-6 exposures reproduced the ketamine effects.Treatment of primary cultures with ketamine increases the expression ofIL-6 mRNA, and injection of IL-6 increased Nox2 expression and activityin brain and in synaptosomes. While the invention is not limited by anyparticular mechanism of action, the invention demonstrates that thefollowing sequence of events can be triggered by sub-anesthetic doses ofNMDA-receptor antagonists:

-   1) NMDA receptor antagonists, in part through inhibition of    NR2A-containing receptors in PV-interneurons, induce disinhibition    of circuitry in the PFC and other cortical regions, leading to    increased glutamate release.-   2) This increased glutamate leads to increased IL-6 and to the    activation of neuronal Nox, and    production.-   3) IL-6 induces Nox subunits, which further increase    production at synaptic sites.-   4) Nox-dependent    mediates oxidation of key ion channels (e.g. redox site on the NMDA    receptor itself), and key enzymes (i.e. serine-racemase) leading to    a secondary hypofunction of cortical circuits.-   5) The initial hypoNMDA state, mediated first by the NMDA-R    antagonist and then by    -dependent inhibition of NMDA-R via its redox site (and possibly    through redox-dependent effects on other synaptic proteins), leads    to a resetting of excitatory transmission. This decreased    glutamatergic transmission is detected by PV-interneurons resulting    in reduced expression of parvalbumin, GAD67, nicotinic receptors,    GAT-1, and thus in a chronically decreased inhibitory tone in    forebrain structures.

This sequence of events (from 1 to 4) appears to be relevant toschizophrenia in its initial phase, when psychotic episodes are morefrequent, but can also lead to sustained dysfunction of inhibitorycircuits, involving PV-interneurons throughout the brain.

While the invention is not limited by any particular mechanism ofaction, the invention demonstrates that increased levels of IL-6 inschizophrenic patients are in part responsible for inducing a mildinflammatory state in the CNS (brain) which activates superoxide and/orhydrogen peroxide production by any member of the NADPH oxidase enzymefamily (“Nox”), e.g., NADPH oxidase-2, to cause dysfunction and thewell-described loss of GABAergic phenotype of PV-interneurons,specifically. This pathway appears to also underlie the reducedantioxidant capacity and decreased glutathione content that has beenconsistently observed in schizophrenic subjects.

Thus, this invention provides prophylactic and ameliorative treatmentsaddressing the basic pathobiology and pathophysiology of CNSinflammation, schizophrenia, delirium, depression, psychosis, traumaticwar neurosis, post traumatic stress disorder (PTSD) and post-traumaticstress syndrome (PTSS), Amyotrophic Lateral Sclerosis (ALS, or LouGehrig's Disease), and/or Multiple Sclerosis (MS), frailty syndrome(FS), aging, and cognitive, learning or memory impairments resultingtherefrom; and the invention provides compositions and methodscomprising use of anti-IL-6 (e.g., tociluzimab, a clinically-approvedmAb for IL-6R), anti-NFκB (in clinical development for cancer) andbrain-targeted anti-Nox compositions, including anti-Nox inhibitorynucleic acid sequences and anti-Nox antibodies. These compositions andmethods of the invention can be optimized using model systems andpatients to determine optimal formulations and dosage and treatmentregimens for ameliorating or preventing frailty syndrome (FS), aging,psychosis, schizophrenia, depression, delirium, CNS inflammation and thelike, and cognitive, learning or memory impairments resulting therefrom,associated with these diseases and conditions. Thus, in alternativeembodiments, compositions and methods of the invention target NFkB,IL-6, IL-6-R or any member of the NADPH oxidase enzyme family to providenovel non-neurotransmitter-based therapeutic treatments for frailtysyndrome (FS), aging, psychosis, schizophrenia, depression, delirium,CNS inflammation, drug-induced psychosis, psychotic features associatedwith frailty syndrome (FS), aging, depression and/or dementias;traumatic war neurosis, post traumatic stress disorder (PTSD) and/orpost-traumatic stress syndrome (PTSS), and/or Amyotrophic LateralSclerosis (ALS, or Lou Gehrig's Disease) and/or Multiple Sclerosis (MS),and the like, and cognitive, learning or memory impairments resultingtherefrom.

In alternative embodiments, compositions and methods of the inventionare used to ameliorate (including to slow, reverse or abate) theincreasing vulnerability to neurodegenerative disorders associated withfrailty syndrome (FS), aging, pathologies, diseases (includinginfections) and conditions associated with an increased amount of CNSinflammation and/or CNS oxidative stress, including Alzheimer's disease,Lewy Body Disease, Parkinson's Disease, Huntington's Disease,Multi-infarct dementia, senile dementia or Frontotemporal Dementia,PTSD. PTSS, Amyotrophic Lateral Sclerosis (ALS, or Lou Gehrig'sDisease), and/or Multiple Sclerosis (MS), and the like, and cognitive,learning or memory impairments resulting therefrom. See Examples 3 and4, below.

Generating and Manipulating Nucleic Acids

In alternative aspects, the invention provides, e.g., isolated,synthetic and/or recombinant nucleic acids encoding inhibitory nucleicacids (e.g., siRNA, microRNA, antisense) that can inhibit the expressionof genes or messages of any member of the NADPH oxidase, particularlyNADPH oxidase in brain cells such as parvalbumin-positive GABA-ergicinterneurons in the cortex. The nucleic acids of the invention can bemade, isolated and/or manipulated by, e.g., cloning and expression ofcDNA libraries, amplification of message or genomic DNA by PCR, and thelike.

The nucleic acids used to practice this invention, whether RNA, iRNA,antisense nucleic acid, cDNA, genomic DNA, vectors, viruses or hybridsthereof, may be isolated from a variety of sources, geneticallyengineered, amplified, and/or expressed/generated recombinantly.Recombinant polypeptides (e.g., anti-NFkB, anti-IL-6, anti-IL-6-R,anti-Nox antibodies used to practice this invention) generated fromthese nucleic acids can be individually isolated or cloned and testedfor a desired activity. Any recombinant expression system can be used,including bacterial, fungal, mammalian, yeast, insect or plant cellexpression systems.

Alternatively, these nucleic acids can be synthesized in vitro bywell-known chemical synthesis techniques, as described in, e.g., Adams(1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res.25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers(1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90;Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett.22:1859; U.S. Pat. No. 4,458,066.

Techniques for the manipulation of nucleic acids, such as, e.g.,subcloning, labeling probes (e.g., random-primer labeling using Klenowpolymerase, nick translation, amplification), sequencing, hybridizationand the like are well described in the scientific and patent literature,see, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2NDED.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc.,New York (1997); LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULARBIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory andNucleic Acid Preparation, Tijssen, ed. Elsevier. N.Y. (1993).

Another useful means of obtaining and manipulating nucleic acids used topractice the methods of the invention is to clone from genomic samples,and, if desired, screen and re-clone inserts isolated or amplified from,e.g., genomic clones or cDNA clones. Sources of nucleic acid used in themethods of the invention include genomic or cDNA libraries contained in,e.g., mammalian artificial chromosomes (MACs), see, e.g., U.S. Pat. Nos.5,721,118; 6,025,155; human artificial chromosomes, see, e.g., Rosenfeld(1997) Nat. Genet. 15:333-335; yeast artificial chromosomes (YAC);bacterial artificial chromosomes (BAC); P1 artificial chromosomes, see,e.g., Woon (1998) Genomics 50:306-316; P1-derived vectors (PACs), see,e.g., Kern (1997) Biotechniques 23:120-124; cosmids, recombinantviruses, phages or plasmids.

The invention provides and uses fusion proteins and nucleic acidsencoding them. Any polypeptide used to practice this invention (e.g., anantibody inhibitory to Nox, NFkB, IL-6 or IL-6-R activity) can be fusedto a heterologous peptide or polypeptide, such as a peptide fortargeting an inhibitory compound used to practice this invention tobrain cells such as parvalbumin-positive GABA-ergic interneurons in thecortex; or the heterologous peptide or polypeptide can be an N-terminalidentification peptide which imparts a desired characteristic, such asfluorescent detection, increased stability and/or simplifiedpurification. Peptides and polypeptides used to practice this inventioncan also be synthesized and expressed as fusion proteins with one ormore additional domains linked thereto for, e.g., producing a moreimmunogenic peptide, to more readily isolate a recombinantly synthesizedpeptide, to identify and isolate antibodies and antibody-expressing Bcells, and the like. Detection and purification facilitating domainsinclude, e.g., metal chelating peptides such as polyhistidine tracts andhistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp, Seattle Wash.). The inclusion of acleavable linker sequences such as Factor Xa or enterokinase(Invitrogen, San Diego Calif.) between a purification domain and themotif-comprising peptide or polypeptide to facilitate purification. Forexample, an expression vector can include an epitope-encoding nucleicacid sequence linked to six histidine residues followed by a thioredoxinand an enterokinase cleavage site (see e.g., Williams (1995)Biochemistry 34:1787-1797; Dobeli (1998) Protein Expr. Purif.12:404-414). The histidine residues facilitate detection andpurification while the enterokinase cleavage site provides a means forpurifying the epitope from the remainder of the fusion protein.Technology pertaining to vectors encoding fusion proteins andapplication of fusion proteins are well described in the scientific andpatent literature, see e.g., Kroll (1993) DNA Cell. Biol., 12:441-53.

Nucleic acids or nucleic acid sequences used to practice this inventioncan be an oligonucleotide, nucleotide, polynucleotide, or to a fragmentof any of these, to DNA or RNA of genomic or synthetic origin which maybe single-stranded or double-stranded and may represent a sense orantisense strand, to peptide nucleic acid (PNA), or to any DNA-like orRNA-like material, natural or synthetic in origin. Compounds use topractice this invention include “nucleic acids” or “nucleic acidsequences” including oligonucleotide, nucleotide, polynucleotide, or anyfragment of any of these; and include DNA or RNA (e.g., mRNA, rRNA,tRNA, iRNA) of genomic or synthetic origin which may be single-strandedor double-stranded; and can be a sense or antisense strand, or a peptidenucleic acid (PNA), or any DNA-like or RNA-like material, natural orsynthetic in origin, including, e.g., iRNA, ribonucleoproteins (e.g.,e.g., double stranded iRNAs, e.g., iRNPs). Compounds use to practicethis invention include nucleic acids, i.e., oligonucleotides, containingknown analogues of natural nucleotides. Compounds use to practice thisinvention include nucleic-acid-like structures with synthetic backbones,see e.g., Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197;Strauss-Soukup (1997) Biochemistry 36:8692-8698; Samstag (1996)Antisense Nucleic Acid Drug Dev 6:153-156. Compounds use to practicethis invention include “oligonucleotides” including a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandsthat may be chemically synthesized. Compounds use to practice thisinvention include synthetic oligonucleotides having no 5′ phosphate, andthus will not ligate to another oligonucleotide without adding aphosphate with an ATP in the presence of a kinase. A syntheticoligonucleotide can ligate to a fragment that has not beendephosphorylated.

In alternative aspects, compounds used to practice this inventioninclude genes or any segment of DNA involved in producing a polypeptidechain (e.g., an anti-IL-6 antibody); it can include regions precedingand following the coding region (leader and trailer) as well as, whereapplicable, intervening sequences (introns) between individual codingsegments (exons). “Operably linked” can refer to a functionalrelationship between two or more nucleic acid (e.g., DNA) segments. Inalternative aspects, it can refer to the functional relationship oftranscriptional regulatory sequence to a transcribed sequence. Forexample, a promoter can be operably linked to a coding sequence, such asa nucleic acid used to practice this invention, if it stimulates ormodulates the transcription of the coding sequence in an appropriatehost cell or other expression system. In alternative aspects, promotertranscriptional regulatory sequences can be operably linked to atranscribed sequence where they can be physically contiguous to thetranscribed sequence, i.e., they can be cis-acting. In alternativeaspects, transcriptional regulatory sequences, such as enhancers, neednot be physically contiguous or located in close proximity to the codingsequences whose transcription they enhance.

In alternative aspects, the invention comprises use of “expressioncassettes” comprising a nucleotide sequence used to practice thisinvention, which can be capable of affecting expression of the nucleicacid. e.g., a structural gene or a transcript (i.e., encoding NFkB,interleukin-6 (IL-6) or any member of the NADPH oxidase) in a hostcompatible with such sequences. Expression cassettes can include atleast a promoter operably linked with the polypeptide coding sequence orinhibitory sequence; and, in one aspect, with other sequences, e.g.,transcription termination signals. Additional factors necessary orhelpful in effecting expression may also be used, e.g., enhancers.

In alternative aspects, expression cassettes used to practice thisinvention also include plasmids, expression vectors, recombinantviruses, any form of recombinant “naked DNA” vector, and the like. Inalternative aspects, a “vector” used to practice this invention cancomprise a nucleic acid that can infect, transfect, transiently orpermanently transduce a cell. In alternative aspects, a vector used topractice this invention can be a naked nucleic acid, or a nucleic acidcomplexed with protein or lipid. In alternative aspects, vectors used topractice this invention can comprise viral or bacterial nucleic acidsand/or proteins, and/or membranes (e.g., a cell membrane, a viral lipidenvelope, etc.). In alternative aspects, vectors used to practice thisinvention can include, but are not limited to replicons (e.g., RNAreplicons, bacteriophages) to which fragments of DNA may be attached andbecome replicated. Vectors thus include, but are not limited to RNA,autonomous self-replicating circular or linear DNA or RNA (e.g.,plasmids, viruses, and the like, see, e.g., U.S. Pat. No. 5,217,879),and can include both the expression and non-expression plasmids. Inalternative aspects, the vector used to practice this invention can bestably replicated by the cells during mitosis as an autonomousstructure, or can be incorporated within the host's genome.

In alternative aspects, “promoters” used to practice this inventioninclude all sequences capable of driving transcription of a codingsequence in a cell, e.g., a mammalian cell such as a brain cell. Thus,promoters used in the constructs of the invention include cis-actingtranscriptional control elements and regulatory sequences that areinvolved in regulating or modulating the timing and/or rate oftranscription of a gene. For example, a promoter used to practice thisinvention can be a cis-acting transcriptional control element, includingan enhancer, a promoter, a transcription terminator, an origin ofreplication, a chromosomal integration sequence, 5′ and 3′ untranslatedregions, or an intronic sequence, which are involved in transcriptionalregulation. These cis-acting sequences typically interact with proteinsor other biomolecules to carry out (turn on/off, regulate, modulate,etc.) transcription.

“Constitutive” promoters used to practice this invention can be thosethat drive expression continuously under most environmental conditionsand states of development or cell differentiation. “Inducible” or“regulatable” promoters used to practice this invention can directexpression of the nucleic acid of the invention under the influence ofenvironmental conditions or developmental conditions. Examples ofenvironmental conditions that may affect transcription by induciblepromoters used to practice this invention include the presence of aninducing factor administered to a subject. “Tissue-specific” promotersused to practice this invention can be transcriptional control elementsthat are only active in particular cells or tissues or organs, e.g., inbrain cells. Tissue-specific regulation may be achieved by certainintrinsic factors that ensure that genes encoding proteins specific to agiven tissue, e.g., brain, are expressed.

Antisense Inhibitory Nucleic Acid Molecules

In alternative embodiments, the invention provides antisense inhibitorynucleic acid molecules capable of decreasing or inhibiting expression ofNFkB, IL-6, IL-6-R, or any member of the NADPH oxidase enzyme family oneither a transcriptional and/or translational level. Naturally occurringor synthetic nucleic acids can be used as antisense oligonucleotides.The antisense oligonucleotides can be of any length; for example, inalternative aspects, the antisense oligonucleotides are between about 5to 100, about 10 to 80, about 15 to 60, about 18 to 40). The optimallength can be determined by routine screening. The antisenseoligonucleotides can be present at any concentration. The optimalconcentration can be determined by routine screening. A wide variety ofsynthetic, non-naturally occurring nucleotide and nucleic acid analoguesare known which can address this potential problem. For example, peptidenucleic acids (PNAs) containing non-ionic backbones, such asN-(2-aminoethyl) glycine units can be used. Antisense oligonucleotideshaving phosphorothioate linkages can also be used, as described in WO97/03211; WO 96/39154; Mata (1997) Toxicol Appl Pharmacol 144:189-197;Antisense Therapeutics, ed. Agrawal (Humana Press, Totowa, N.J., 1996).Antisense oligonucleotides having synthetic DNA backbone analoguesprovided by the invention can also include phosphoro-dithioate,methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate,3′-thioacetal, methylene(methylimino), 3′-N-carbamate, and morpholinocarbamate nucleic acids.

RNA Interference (RNAi)

In one aspect, the invention provides an RNA inhibitory molecule, aso-called “RNAi” molecule, comprising a sequence capable of decreasingor inhibiting expression of NFkB, IL-6, IL-6-R, or any member of theNADPH oxidase enzyme family on either a transcriptional and/ortranslational level. In one aspect, the RNAi molecule comprises adouble-stranded RNA (dsRNA) molecule. The RNAi molecule can comprise adouble-stranded RNA (dsRNA) molecule, e.g., siRNA, miRNA (microRNA)and/or short hairpin RNA (shRNA) molecules. The RNAi molecule, e.g.,siRNA (small inhibitory RNA) can inhibit expression of a gene of anymember of the NADPH oxidase enzyme family, and/or miRNA (micro RNA) toinhibit translation of NFkB, IL-6, IL-6-R, or a NADPH oxidase gene.

In alternative aspects, the RNAi is about 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25 or more duplex nucleotides in length.While the invention is not limited by any particular mechanism ofaction, the RNAi can enter a cell and cause the degradation of asingle-stranded RNA (ssRNA) of similar or identical sequences, includingendogenous mRNAs. When a cell is exposed to double-stranded RNA (dsRNA),mRNA from the homologous gene is selectively degraded by a processcalled RNA interference (RNAi). A possible basic mechanism behind RNAi,e.g., siRNA for inhibiting transcription and/or miRNA to inhibittranslation, is the breaking of a double-stranded RNA (dsRNA) matching aspecific gene sequence into short pieces called short interfering RNA,which trigger the degradation of mRNA that matches its sequence. In oneaspect, the RNAi's of the invention are used in gene-silencingtherapeutics, see, e.g., Shuey (2002) Drug Discov. Today 7:1040-1046. Inone aspect, the invention provides methods to selectively degrade RNAusing the RNAi's of the invention. The process may be practiced invitro, ex vivo or in vivo. In one aspect, the RNAi molecules of theinvention can be used to generate a loss-of-function mutation in a cell,an plant tissue or organ or seed, or a plant.

In one aspect, intracellular introduction of the RNAi (e.g., miRNA orsiRNA) is by internalization of a target cell specific ligand bonded toan RNA binding protein comprising an RNAi (e.g., microRNA) is adsorbed.The ligand is specific to a unique target cell surface antigen. Theligand can be spontaneously internalized after binding to the cellsurface antigen. If the unique cell surface antigen is not naturallyinternalized after binding to its ligand, internalization can bepromoted by the incorporation of an arginine-rich peptide, or othermembrane permeable peptide, into the structure of the ligand or RNAbinding protein or attachment of such a peptide to the ligand or RNAbinding protein. See, e.g., U.S. Patent App. Pub. Nos. 20060030003;20060025361; 20060019286; 20060019258. In one aspect, the inventionprovides lipid-based formulations for delivering. e.g., introducingnucleic acids of the invention as nucleic acid-lipid particlescomprising an RNAi molecule to a cell, see e.g., U.S. Patent App. Pub.No. 20060008910.

Methods for making and using RNAi molecules, e.g., siRNA and/or miRNA,for selectively degrade RNA are well known in the art, see, e.g., U.S.Pat. Nos. 6,506,559; 6,511,824; 6,515,109; 6,489,127.

Methods for making expression constructs, e.g., vectors or plasmids,from which an inhibitory polynucleotide (e.g., a duplex siRNA of theinvention) is transcribed are well known and routine. A regulatoryregion (e.g., promoter, enhancer, silencer, splice donor, acceptor,etc.) can be used to transcribe an RNA strand or RNA strands of aninhibitory polynucleotide from an expression construct. When making aduplex siRNA inhibitory molecule, the sense and antisense strands of thetargeted portion of the targeted IRES can be transcribed as two separateRNA strands that will anneal together, or as a single RNA strand thatwill form a hairpin loop and anneal with itself. For example, aconstruct targeting a portion of a gene, e.g., an NADPH oxidase enzymecoding sequence or transcriptional activation sequence, is insertedbetween two promoters (e.g., mammalian, viral, human, tissue specific,constitutive or other type of promoter) such that transcription occursbidirectionally and will result in complementary RNA strands that maysubsequently anneal to form an inhibitory siRNA of the invention.

Alternatively, a targeted portion of gene, coding sequence, promoter ortranscript can be designed as a first and second antisense bindingregion together on a single expression vector; for example, comprising afirst coding region of a targeted NADPH oxidase gene in senseorientation relative to its controlling promoter, and wherein the secondcoding region of a NADPH oxidase gene is in antisense orientationrelative to its controlling promoter. If transcription of the sense andantisense coding regions of the targeted portion of the targeted geneoccurs from two separate promoters, the result may be two separate RNAstrands that may subsequently anneal to form a gene inhibitory siRNA,e.g., a NADPH oxidase gene-inhibitory siRNA used to practice thisinvention.

In another aspect, transcription of the sense and antisense targetedportion of the targeted NADPH oxidase gene is controlled by a singlepromoter, and the resulting transcript will be a single hairpin RNAstrand that is self-complementary, i.e., forms a duplex by folding backon itself to create a NADPH oxidase gene-inhibitory siRNA molecule. Inthis configuration, a spacer, e.g., of nucleotides, between the senseand antisense coding regions of the targeted portion of the targetedNADPH oxidase gene can improve the ability of the single strand RNA toform a hairpin loop, wherein the hairpin loop comprises the spacer. Inones embodiment, the spacer comprises a length of nucleotides of betweenabout 5 to 50 nucleotides. In one aspect, the sense and antisense codingregions of the siRNA can each be on a separate expression vector andunder the control of its own promoter.

Inhibitory Ribozymes

The invention provides ribozymes capable of binding and inhibiting genesand/or messages (transcripts) from NFkB, IL-6, IL-6-R, or any member ofthe NADPH oxidase enzyme family. These ribozymes can inhibit NADPHoxidase gene activity by, e.g., targeting a genomic DNA or an mRNA (amessage, a transcript). Strategies for designing ribozymes and selectinga NFkB-, IL-6-, IL-6-R-, or NADPH oxidase gene-specific antisensesequence for targeting are well described in the scientific and patentliterature, and the skilled artisan can design such ribozymes using thenovel reagents of the invention. Ribozymes act by binding to a targetRNA through the target RNA binding portion of a ribozyme which is heldin close proximity to an enzymatic portion of the RNA that cleaves thetarget RNA. Thus, the ribozyme recognizes and binds a target RNA throughcomplementary base-pairing, and once bound to the correct site, actsenzymatically to cleave and inactivate the target RNA. Cleavage of atarget RNA in such a manner will destroy its ability to direct synthesisof an encoded protein if the cleavage occurs in the coding sequence.After a ribozyme has bound and cleaved its RNA target, it can bereleased from that RNA to bind and cleave new targets repeatedly.

Polypeptides and Peptides

In alternative embodiments, the invention provides polypeptides andpeptides to inhibit or decrease the amount of active NFkB, IL-6, IL-6-R,any member of the NADPH oxidase enzyme family, and/or superoxide and/orhydrogen peroxide production by inhibiting or decreasing the activity ofthe enzyme NADPH oxidase and/or IL-6 or IL-6 receptor (IL-6-R),including antibodies or peptides for inhibiting IL-6, IL-6-R and/orNADPH oxidase activity in the brain. In alternative embodiments, NFkB,IL-6, IL-6-R and/or NADPH oxidase inhibitors used to practice thisinvention are proteins or antibodies that specifically bind to andinhibit the activity of NFkB, IL-6, IL-6-R and/or NADPH oxidase enzymes.

Polypeptides and peptides used to practice the invention can be isolatedfrom natural sources, be synthetic, or be recombinantly generatedpolypeptides. Peptides and proteins can be recombinantly expressed invitro or in vivo. The peptides and polypeptides used to practice theinvention can be made and isolated using any method known in the art.Polypeptide and peptides used to practice the invention can also besynthesized, whole or in part, using chemical methods well known in theart. See e.g., Caruthers (1980) Nucleic Acids Res. Symp. Ser. 215-223;Horn (1980) Nucleic Acids Res. Symp. Ser. 225-232; Banga, A. K.,Therapeutic Peptides and Proteins, Formulation, Processing and DeliverySystems (1995) Technomic Publishing Co., Lancaster, Pa. For example,peptide synthesis can be performed using various solid-phase techniques(see e.g., Roberge (1995) Science 269:202; Merrifield (1997) MethodsEnzymol. 289:3-13) including any automated polypeptide synthesis processknown in the art.

The peptides and polypeptides used to practice the invention can also beglycosylated. The glycosylation can be added post-translationally eitherchemically or by cellular biosynthetic mechanisms, wherein the laterincorporates the use of known glycosylation motifs, which can be nativeto the sequence or can be added as a peptide or added in the nucleicacid coding sequence. The glycosylation can be O-linked or N-linked.

In alternative embodiments, compositions used to practice the inventioncomprise an oligopeptide, peptide, polypeptide, or protein sequence, orto a fragment, portion, or subunit of any of these and to naturallyoccurring or synthetic molecules. In alternative aspects, polypeptidesused to practice the invention comprise amino acids joined to each otherby peptide bonds or modified peptide bonds and may comprise modifiedamino acids other than the 20 gene-encoded amino acids. The polypeptidesmay be modified by either natural processes, such as post-translationalprocessing, or by chemical modification techniques that are well knownin the art. Modifications can occur anywhere in the polypeptide,including the peptide backbone, the amino acid side-chains and the aminoor carboxyl termini. It will be appreciated that the same type ofmodification may be present in the same or varying degrees at severalsites in a given polypeptide.

In alternative embodiments, a polypeptide used to practice the inventioncan have many types of modifications, e.g., modifications includingacetylation, acylation, ADP-ribosylation, amidation, covalent attachmentof flavin, covalent attachment of a heme moiety, covalent attachment ofa nucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of a phosphatidylinositol,cross-linking cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cysteine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation. GPIanchor formation, hydroxylation, iodination, methylation,myristolyation, oxidation, pegylation, phosphorylation, prenylation,racemization, selenoylation, sulfation and transfer-RNA mediatedaddition of amino acids to protein such as arginylation. See forexample, Creighton, T. E., Proteins—Structure and Molecular Properties2nd Ed., W.H. Freeman and Company, New York (1993); PosttranslationalCovalent Modification of Proteins, B. C. Johnson, Ed., Academic Press,New York. pp. 1-12 (1983)).

In alternative embodiments, peptides and polypeptides used to practicethe invention can comprise any “mimetic” and/or “peptidomimetic” form.In alternative embodiments, peptides and polypeptides used to practicethe invention can comprise synthetic chemical compounds which havesubstantially the same structural and/or functional characteristics ofnatural polypeptides. The mimetic used to practice the invention can beeither entirely composed of synthetic, non-natural analogues of aminoacids, or, is a chimeric molecule of partly natural peptide amino acidsand partly non-natural analogs of amino acids. The mimetic can alsoincorporate any amount of natural amino acid conservative substitutionsas long as such substitutions also do not substantially alter themimetic's structure and/or activity. Routine experimentation willdetermine whether a mimetic is effective for practicing the invention;e.g., a mimetic composition is effective if it has an NFkB, IL-6, IL-6-Rand/or NADPH oxidase (Nox) inhibitory activity. Methodologies detailedherein and others known to persons skilled in the art may be used toselect or guide one to choose effective mimetic for practicing thecompositions and/or methods of this invention.

Polypeptide mimetic compositions for practicing the invention cancomprise any combination of non-natural structural components. Inalternative aspects, mimetic compositions for practicing the inventioncan comprise one or all of the following three structural groups: a)residue linkage groups other than the natural amide bond (“peptidebond”) linkages; b) non-natural residues in place of naturally occurringamino acid residues; or c) residues which induce secondary structuralmimicry, i.e., to induce or stabilize a secondary structure, e.g., abeta turn, gamma turn, beta sheet, alpha helix conformation, and thelike. For example, a polypeptide can be characterized as a mimetic whenall or some of its residues are joined by chemical means other thannatural peptide bonds. Individual peptidomimetic residues can be joinedby peptide bonds, other chemical bonds or coupling means, such as, e.g.,glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides,N,N′-dicyclohexylcarbodiimide (DCC) or N,N′-diisopropylcarbodiimide(DIC). Linking groups that can be an alternative to the traditionalamide bond (“peptide bond”) linkages include, e.g., ketomethylene (e.g.,—C(═O)—CH₂— for —C(═O)—NH—), aminomethylene (CH₂—NH), ethylene, olefin(CH═CH), ether (CH₂—O), thioether (CH₂—S), tetrazole (CN₄—), thiazole,retroamide, thioamide, or ester (see, e.g., Spatola (1983) in Chemistryand Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp267-357, “Peptide Backbone Modifications,” Marcell Dekker, NY). Apolypeptide can also be characterized as a mimetic by containing all orsome non-natural residues in place of naturally occurring amino acidresidues. Non-natural residues are well described in the scientific andpatent literature; a few exemplary non-natural compositions useful asmimetics of natural amino acid residues and guidelines are describedbelow. Mimetics of aromatic amino acids can be generated by replacingby, e.g., D- or L-naphylalanine; D- or L-phenylglycine; D- or L-2thieneylalanine; D- or L-1,-2, 3-, or 4-pyreneylalanine; D- or L-3thieneylalanine; D- or L-(2-pyridinyl)-alanine; D- orL-(3-pyridinyl)-alanine; D- or L-(2-pyrazinyl)-alanine; D- orL-(4-isopropyl)-phenylglycine; D-(trifluoromethyl)-phenylglycine;D-(trifluoromethyl)-phenylalanine; D-p-fluoro-phenylalanine D- orL-p-biphenylphenylalanine; D- or L-p-methoxy-biphenylphenylalanine; D-or L-2-indole(alkyl)alanines; and, D- or L-alkylainines, where alkyl canbe substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl,pentyl, isopropyl, iso-butyl, sec-isotyl, iso-pentyl, or a non-acidicamino acids. Aromatic rings of a non-natural amino acid include, e.g.,thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl,pyrrolyl, and pyridyl aromatic rings.

Mimetics of acidic amino acids can be generated by substitution by,e.g., non-carboxylate amino acids while maintaining a negative charge;(phosphono)alanine; sulfated threonine. Carboxyl side groups (e.g.,aspartyl or glutamyl) can also be selectively modified by reaction withcarbodiimides (R′—N—C—N—R′) such as, e.g.,1-cyclohexyl-3(2-morpholinyl-(4-ethyl) carbodiimide or1-ethyl-3(4-azonia-4,4-dimetholpentyl) carbodiimide. Aspartyl orglutamyl can also be converted to asparaginyl and glutaminyl residues byreaction with ammonium ions. Mimetics of basic amino acids can begenerated by substitution with, e.g., (in addition to lysine andarginine) the amino acids omithine, citrulline, or (guanidino)-aceticacid, or (guanidino)alkyl-acetic acid, where alkyl is defined above.Nitrile derivative (e.g., containing the CN-moiety in place of COOH) canbe substituted for asparagine or glutamine. Asparaginyl and glutaminylresidues can be deaminated to the corresponding aspartyl or glutamylresidues. Arginine residue mimetics can be generated by reacting arginylwith, e.g., one or more conventional reagents, including. e.g.,phenylglyoxal, 2,3-butanedione, 1,2-cyclo-hexanedione, or ninhydrin,e.g., under alkaline conditions. Tyrosine residue mimetics can begenerated by reacting tyrosyl with, e.g., aromatic diazonium compoundsor tetranitromethane. N-acetylimidizol and tetranitromethane can be usedto form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.Cysteine residue mimetics can be generated by reacting cysteinylresidues with, e.g., alpha-haloacetates such as 2-chloroacetic acid orchloroacetamide and corresponding amines; to give carboxymethyl orcarboxyamidomethyl derivatives. Cysteine residue mimetics can also begenerated by reacting cysteinyl residues with, e.g.,bromo-trifluoroacetone, alpha-bromo-beta-(5-imidozoyl) propionic acid;chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide;methyl 2-pyridyl disulfide; p-chloromercuribenzoate; 2-chloromercuri-4nitrophenol; or, chloro-7-nitrobenzo-oxa-1,3-diazole. Lysine mimeticscan be generated (and amino terminal residues can be altered) byreacting lysinyl with, e.g., succinic or other carboxylic acidanhydrides. Lysine and other alpha-amino-containing residue mimetics canalso be generated by reaction with imidoesters, such as methylpicolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride,trinitro-benzenesulfonic acid, O-methylisourea, 2,4, pentanedione, andtransamidase-catalyzed reactions with glyoxylate. Mimetics of methioninecan be generated by reaction with, e.g., methionine sulfoxide. Mimeticsof proline include, e.g., pipecolic acid, thiazolidine carboxylic acid,3- or 4-hydroxy proline, dehydroproline, 3- or 4-methylproline, or3,3,-dimethylproline. Histidine residue mimetics can be generated byreacting histidyl with, e.g., diethylprocarbonate or para-bromophenacylbromide. Other mimetics that can be used include, e.g., those generatedby hydroxylation of proline and lysine; phosphorylation of the hydroxylgroups of seryl or threonyl residues; methylation of the alpha-aminogroups of lysine, arginine and histidine; acetylation of the N-terminalamine; methylation of main chain amide residues or substitution withN-methyl amino acids; or amidation of C-terminal carboxyl groups.

Polypeptides used to practice this invention can comprise signalsequences, i.e., leader sequences, e.g., for secreting a recombinantantibody or inhibitory polypeptide used to practice the invention from aproduction host cell.

Antibodies, Therapeutic and Humanized Antibodies

In alternative embodiments, the invention uses isolated, synthetic orrecombinant antibodies that specifically bind to and inhibit an IL-6 orIL-6 receptor, or to NADPH oxidase; for example, practicing theinvention can comprise use of a therapeutic monoclonal antibodyinhibitory to NFkB, NADPH oxidase, IL-6 or IL-6 receptor activity (wherethe antibody acts as a specific antagonist (is receptor-inhibiting) forIL-6 receptors).

In alternative aspects, an antibody for practicing the invention cancomprise a peptide or polypeptide derived from, modeled after orsubstantially encoded by an immunoglobulin gene or immunoglobulin genes,or fragments thereof, capable of specifically binding an antigen orepitope, see, e.g. Fundamental Immunology, Third Edition, W. E. Paul,ed., Raven Press, N.Y. (1993); Wilson (1994) J. Immunol. Methods175:267-273; Yarmush (1992) J. Biochem. Biophys. Methods 25:85-97. Inalternative aspects, an antibody for practicing the invention includesantigen-binding portions, i.e., “antigen binding sites,” (e.g.,fragments, subsequences, complementarity determining regions (CDRs))that retain capacity to bind antigen, including (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) aF(ab′)2 fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CHI domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546), which consists of a VH domain;and (vi) an isolated complementarity determining region (CDR). Singlechain antibodies are also included by reference in the term “antibody.”

Methods of immunization, producing and isolating antibodies (polyclonaland monoclonal) are known to those of skill in the art and described inthe scientific and patent literature, see, e.g., Coligan, CURRENTPROTOCOLS IN IMMUNOLOGY, Wiley/Greene, NY (1991); Stites (eds.) BASICAND CLINICAL IMMUNOLOGY (7th ed.) Lange Medical Publications, Los Altos,Calif. (“Stites”); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES ANDPRACTICE (2d ed.) Academic Press, New York, N.Y. (1986); Kohler (1975)Nature 256:495; Harlow (1988) ANTIBODIES, A LABORATORY MANUAL, ColdSpring Harbor Publications, New York. Antibodies also can be generatedin vitro, e.g., using recombinant antibody binding site expressing phagedisplay libraries, in addition to the traditional in vivo methods usinganimals. See, e.g., Hoogenboom (1997) Trends Biotechnol. 15:62-70; Katz(1997) Annu. Rev. Biophys. Biomol. Struct. 26:27-45.

In alternative embodiments, the invention uses “humanized” antibodies,including forms of non-human (e.g., murine) antibodies that are chimericantibodies comprising minimal sequence (e.g., the antigen bindingfragment) derived from non-human immunoglobulin. In alternativeembodiments, humanized antibodies are human immunoglobulins in whichresidues from a hypervariable region (HVR) of a recipient (e.g., a humanantibody sequence) are replaced by residues from a hypervariable region(HVR) of a non-human species (donor antibody) such as mouse, rat, rabbitor nonhuman primate having the desired specificity, affinity, andcapacity. In alternative embodiments, framework region (FR) residues ofthe human immunoglobulin are replaced by corresponding non-humanresidues to improve antigen binding affinity.

In alternative embodiments, humanized antibodies may comprise residuesthat are not found in the recipient antibody or the donor antibody.These modifications may be made to improve antibody affinity orfunctional activity. In alternative embodiments, the humanized antibodycan comprise substantially all of at least one, and typically two,variable domains, in which all or substantially all of the hypervariableregions correspond to those of a non-human immunoglobulin and all orsubstantially all of Ab framework regions are those of a humanimmunoglobulin sequence.

In alternative embodiments, a humanized antibody used to practice thisinvention can comprise at least a portion of an immunoglobulin constantregion (Fc), typically that of or derived from a human immunoglobulin.

However, in alternative embodiments, completely human antibodies alsocan be used to practice this invention, including human antibodiescomprising amino acid sequence which corresponds to that of an antibodyproduced by a human. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen bindingresidues.

In alternative embodiments, antibodies used to practice this inventioncomprise “affinity matured” antibodies, e.g., antibodies comprising withone or more alterations in one or more hypervariable regions whichresult in an improvement in the affinity of the antibody for antigen;e.g., NFkB, interleukin-6 (IL-6), IL-6-R and/or an NADPH oxidase (Nox)enzyme family member, compared to a parent antibody which does notpossess those alteration(s). In alternative embodiments, antibodies usedto practice this invention are matured antibodies having nanomolar oreven picomolar affinities for the target antigen, e.g., NFkB,interleukin-6 (IL-6), NADPH oxidase (Nox) enzyme. Affinity maturedantibodies can be produced by procedures known in the art.

Tocilizumab

In one embodiment, therapeutic monoclonal antibodies against any and/orall member(s) of the IL-6-R family are used to practice this invention;and in one aspect, the antibody acts as a specific antagonist (isreceptor-inhibiting) for IL-6 receptors, e.g., tocilizumab, or ACTEMRA™(F. Hoffmann-La Roche Ltd, Basel, Switzerland). This invention can usethe known methods for formulating and administering tocilizumab, whichis administered to humans for rheumatoid arthritis; see e.g., Yokota etal. (2008) Lancet 371(9617):998-1006, describing the efficacy and safetyof tocilizumab in children with systemic-onset juvenile idiopathicarthritis, finding that tocilizumab is effective in children with thisdisease. Dosage was given was three doses of tocilizumab 8 mg/kg every 2weeks during a 6-week open-label lead-in phase. In another study adultpatients with rheumatoid arthritis received tocilizumab 8 mg/kg (n=205),tocilizumab 4 mg/kg, see Smolen, et al. (2008) Lancet 371(9617):987-97.

Tocilizumab has a long plasma half-life, so it can be administeredintravenously biweekly or monthly. Published Phase I and 11 clinicaltrials showed that tocilizumab (2, 4, 5, 8 or 10 mg/kg) reducedrheumatoid arthritis disease activity significantly in a dose-dependentmanner. Tocilizumab was generally safe and well tolerated. Some adverseevents such as significant rises in total cholesterol and triglyceridelevels, liver function disorders, decreases in white blood cell counts,diarrhea and infection were observed. The most common adverse eventswere infections, anaphylactic reactions, and hypersensitivity. Insummary, preliminary clinical results showed that tocilizumab iseffective and generally well tolerated in the treatment of IL-6-relatedinflammatory autoimmune diseases. Like other anti-cytokineimmunotherapies, caution and close monitoring for the adverse events,especially infection, are necessary in any clinical trial or treatmentregimen.

In one embodiment, therapeutic monoclonal antibodies against any and/orall member(s) of the NADPH oxidase enzyme family are used to practicethis invention.

NADPH Oxidase Inhibitors

The invention provides compositions and methods to inhibit or decreasethe activity of the enzyme NADPH oxidase (Nox) or any member of theNADPH oxidase subfamily, e.g., Nox1, Nox2, Nox3, Nox4 or Nox5(collectively referred to as “Nox”), as described for example e.g., inU.S. Pat. No. 6,489,149. In alternative embodiments, these NADPH oxidaseinhibitors are synthetic and/or small molecules known in the art, e.g.,including diphenyleneiodonium (DPI), o-methoxycatechols (e.g., apocyninand diapocynin), 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF),4-hydroxy-3′-methoxy-acetophenon, N-Vanillylnonanamide, staurosporineand related compounds, see e.g., U.S. Patent App. Pub. Nos. 20040001818;20060154856. In alternative embodiments, the Nox is inhibited usinganti-Nox antibodies or anti-Nox inhibitory nucleic acids, as describedherein.

In alternative embodiments, NADPH oxidase inhibitors comprising aromaticazines and imines as described e.g., in U.S. Pat. Nos. 5,990,137;5,939,460; or substituted diphenylazomethines as described e.g., in U.S.Pat. No. 4,564,636, can be used to practice this invention.

In alternative embodiments, NADPH oxidase inhibitors comprisingcompounds similar or related to o-methoxycatechol as described e.g., inU.S. Pat. No. 6,090,851, can be used to practice this invention.

In alternative embodiments, the methods of the invention use the NADPHoxidase inhibitor apocynin (4-hydroxy-3-methoxyacetophenone), which is amajor active ingredient from the rhizomes of Picrorhiza kurroa, abotanical plant used as an herbal medicine for treatment of a number ofinflammatory diseases. The bioavailability of apocynin through itsconversion to glycoconjugate but not to diapocynin has been studied anddescribed e.g., by Wang et al. (2008) Phytomedicine 15(6-7):496-503;Epub 2007 Oct. 30. In another aspect, diapocynin is used, noting thatdiapocynin is 13 times more lipophilic than apocynin, as described byLuchtefeld, et al. (2008) J Agric Food Chem. 56(2):301-6. Epub 2007 Dec.20. See also U.S. Pat. No. 6,949,586, describing formulating apocynin;and apocynin has been administered at a dosage of 1.5 mmol/L in drinkingwater in an animal model, see e.g., Elmarakby, et al. (2005)Hypertension 45:283.

NFkB Inhibitors

The invention provides compositions and methods to inhibit or decreasethe activity of NFkB. While the invention is not limited by anyparticular mechanism of action, in one embodiment, inhibiting ordecreasing the activity of NFkB by practicing the compositions and/ormethods of this invention has the effect of decreasing the amount ofsuperoxide or hydrogen peroxide as produced by a member of the NADPHoxidase enzyme family (Nox).

In alternative embodiments, NFkB is inhibited using anti-NFkB antibodiesor anti-NFkB inhibitory nucleic acids, e.g. as described herein or inU.S. Pat. No. 5,591,840. In alternative embodiments, these NFkBinhibitors are synthetic and/or small molecules.

Any NFkB inhibitory molecule can be used, e.g., as described in U.S.Pat. App. Pub. No. 20070031410; or e.g., a therapeutically effectiveamount of a curcumin derivative administering the curcumin derivative asdescribed in U.S. Pat. App. Pub. No. 20060258752. In alternativeembodiments, NFkB is inhibited indirectly, e.g., by inhibiting CARD11nucleic acids as described in U.S. Pat. App. Pub. No. 20040072228; or byincreasing the amount of or activating IκBs, a family of NFkB inhibitoryproteins having an N-terminal regulatory domain followed by six or moreankyrin repeats and a PEST domain near their C terminus, including IκBα,IκBβ, IκBγ, IκBε, and Bcl-3.

In alternative embodiments, SN50, an inhibitor of NF-kB, isadministered. This peptide comprises a nuclear localization sequence(NLS) for NFkB linked to a cell-permeable carrier. SN50 can inhibit NFkBby interfering with its translocation through the nuclear pore. In oneembodiment, the SN50 peptide comprises the sequence:H₂N-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Val-Gln-Arg-Lys-Arg-Gln-Lys-Leu-Met-Pro-OH (SEQ ID NO: 1) (see e.g., Melotti (2001) Gene Therapy8:1436-1442).

Superoxide Dismutase (SOD) Mimetics

The invention provides compositions and methods to mimic the activity ofthe enzyme superoxide dismutase (SOD), wherein the mimetic decreasessuperoxide and/or hydrogen peroxide activity, In one embodiment, the SODmimetic comprises a C60 fullerene, C₃ (tris malonic acid C60) or amalonic acid derivative.

While the invention is not limited by any particular mechanism ofaction, in one embodiment, the C60 fullerenes (e.g., C₃, or tris malonicacid C60) or other malonic acid derivatives act as superoxide dismutasemimetics, thereby augmenting the action of endogenous SOD to decreasethe amount of superoxide, thereby having a cytoprotective effect,including a cytoprotective effect in the CNS. Any fullerene derivatives(e.g., C₃, or tris malonic acid C60) or malonic acid derivatives can beused to practice this invention, including for example a C₃ as describedby e.g., U.S. Pat. No. 6,538,153, Hirsch, et al., describing macrocyclicmalonate compounds, including the tris malonic acid C60; or as describedin U.S. Pat. No. 7,070,810, Hirsch, et al., describing amphiphilicsubstituted fullerenes and fullerenes comprising a fullerene core and afunctional moiety, and methods for making them; or as described by C.Bingel (1993) Chem. Ber. 126:1957, including compositions wherein themalonate is functionalized with a halide atom, or compositions whereester groups are replaced by alkyne groups indialkynylmethanofullerenes. In alternative embodiments, silica coatedC60 fullerene molecules or C60 fullerene-comprising silica coated carbonnanotubes can be used as described in U.S. Pat. App. Pub. No.20080233040; or composites of fullerene nanotubes as described in U.S.Pat. App. Pub. No. 20080224100; or fullerene suspensions as described inU.S. Pat. App. Pub. No. 20080217445; or pharmaceutically acceptablecompositions comprising fullerene molecules dispersed in vesiclescomprising e.g., phosphatidylcholine (PC) phospholipid molecules andnon-PC phospholipid molecules, as described in U.S. Pat. App. Pub. No.20080213352; or synthetically modified fullerene molecules as describedin U.S. Pat. App. Pub. No. 20080213324.

Pharmaceutical Compositions

The invention provides pharmaceutical compositions and methods toinhibit or decrease the amount of active NFkB, IL-6, NADPH oxidaseenzymes, and/or superoxide and/or hydrogen peroxide production, byinhibiting or decreasing the activity of the enzyme NADPH oxidaseenzymes, and/or NFkB, IL-6 or IL-6 receptor (IL-6-R), includingpharmaceutical compositions, e.g., in the manufacture of medicaments forinhibiting NFkB, IL-6, IL-6-R and/or NADPH oxidase enzyme activity inthe brain. These NFkB, IL-6, IL-6-R and/or NADPH oxidase enzymeinhibitors can be proteins, e.g., antibodies that specifically bind toand inhibit the activity of NFkB, IL-6, IL-6-R and/or NADPH oxidaseenzyme, or inhibitory nucleic acids, e.g., RNAi such an iRNA ormicro-inhibitory RNA acting at the transcriptional and/or translationslevel.

The invention provides pharmaceutical compositions comprising compoundsthat act as a superoxide dismutase mimetic to decrease superoxide and/orhydrogen peroxide levels and/or production.

In alternative embodiments, compositions used to practice thisinvention, including NFkB, IL-6, IL-6-R and/or NADPH oxidase enzymeinhibitory compositions, or compositions comprising compounds that actas a superoxide dismutase mimetic to decrease superoxide and/or hydrogenperoxide levels and/or production, are formulated with apharmaceutically acceptable carrier. In alternative embodiments, thepharmaceutical compositions used to practice the invention can beadministered parenterally, topically, orally or by local administration,such as by aerosol or transdermally. The pharmaceutical compositionsused to practice the invention can be formulated in any way and can beadministered in a variety of unit dosage forms depending upon thecondition or disease and the degree of illness, the general medicalcondition of each patient, the resulting preferred method ofadministration and the like. Details on techniques for formulation andadministration are well described in the scientific and patentliterature, see, e.g., the latest edition of Remington's PharmaceuticalSciences, Maack Publishing Co, Easton Pa. (“Remington's”).

Therapeutic agents used to practice the invention, including smallmolecules, inhibitory nucleic acids and antibodies, can be administeredalone or as a component of a pharmaceutical formulation (composition).The compounds may be formulated for administration in any convenient wayfor use in human or veterinary medicine. Wetting agents, emulsifiers andlubricants, such as sodium lauryl sulfate and magnesium stearate, aswell as coloring agents, release agents, coating agents, sweetening,flavoring and perfuming agents, preservatives and antioxidants can alsobe present in the compositions.

Formulations of the compositions used to practice the invention includethose suitable for oral/nasal, topical, parenteral, rectal, and/orintravaginal administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient (includingsmall molecules, inhibitory nucleic acids and antibodies) which can becombined with a carrier material to produce a single dosage form willvary depending upon the host being treated, the particular mode ofadministration. The amount of active ingredient which can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the compound which produces a therapeutic effect.

Pharmaceutical formulations used to practice the invention can beprepared according to any method known to the art for the manufacture ofpharmaceuticals. Such drugs can contain sweetening agents, flavoringagents, coloring agents and preserving agents. A formulation can beadmixtured with nontoxic pharmaceutically acceptable excipients whichare suitable for manufacture. Formulations may comprise one or morediluents, emulsifiers, preservatives, buffers, excipients, etc. and maybe provided in such forms as liquids, powders, emulsions, lyophilizedpowders, sprays, creams, lotions, controlled release formulations,tablets, pills, gels, on patches, in implants, etc.

Pharmaceutical formulations for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art inappropriate and suitable dosages. Such carriers enable thepharmaceuticals to be formulated in unit dosage forms as tablets, pills,powder, dragees, capsules, liquids, lozenges, gels, syrups, slurries,suspensions, etc., suitable for ingestion by the patient. Pharmaceuticalpreparations for oral use can be formulated as a solid excipient,optionally grinding a resulting mixture, and processing the mixture ofgranules, after adding suitable additional compounds, if desired, toobtain tablets or dragee cores. Suitable solid excipients arecarbohydrate or protein fillers include, e.g., sugars, includinglactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice,potato, or other plants; cellulose such as methyl cellulose,hydroxypropylmethyl-cellulose, or sodium carboxy-methylcellulose; andgums including arabic and tragacanth; and proteins, e.g., gelatin andcollagen. Disintegrating or solubilizing agents may be added, such asthe cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a saltthereof, such as sodium alginate.

Dragee cores are provided with suitable coatings such as concentratedsugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound (i.e., dosage).

Pharmaceutical preparations used to practice the invention can also beused orally using, e.g., push-fit capsules made of gelatin, as well assoft, sealed capsules made of gelatin and a coating such as glycerol orsorbitol. Push-fit capsules can contain active agents mixed with afiller or binders such as lactose or starches, lubricants such as talcor magnesium stearate, and, optionally, stabilizers. In soft capsules,the active agents can be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycol withor without stabilizers.

Aqueous suspensions can contain an active agent (e.g., a chimericpolypeptide or peptidomimetic used to practice this invention, e.g., anantibody) in admixture with excipients suitable for the manufacture ofaqueous suspensions. Such excipients include a suspending agent, such assodium carboxymethylcellulose, methylcellulose,hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gumtragacanth and gum acacia, and dispersing or wetting agents such as anaturally occurring phosphatide (e.g., lecithin), a condensation productof an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate),a condensation product of ethylene oxide with a long chain aliphaticalcohol (e.g., heptadecaethylene oxycetanol), a condensation product ofethylene oxide with a partial ester derived from a fatty acid and ahexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensationproduct of ethylene oxide with a partial ester derived from fatty acidand a hexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate).The aqueous suspension can also contain one or more preservatives suchas ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, oneor more flavoring agents and one or more sweetening agents, such assucrose, aspartame or saccharin. Formulations can be adjusted forosmolarity.

Oil-based pharmaceuticals are particularly useful for administration ofthe hydrophobic active agents used to practice the invention. Oil-basedsuspensions can be formulated by suspending an active agent in avegetable oil, such as arachis oil, olive oil, sesame oil or coconutoil, or in a mineral oil such as liquid paraffin; or a mixture of these.See e.g., U.S. Pat. No. 5,716,928 describing using essential oils oressential oil components for increasing bioavailability and reducinginter- and intra-individual variability of orally administeredhydrophobic pharmaceutical compounds (see also U.S. Pat. No. 5,858,401).The oil suspensions can contain a thickening agent, such as beeswax,hard paraffin or cetyl alcohol. Sweetening agents can be added toprovide a palatable oral preparation, such as glycerol, sorbitol orsucrose. These formulations can be preserved by the addition of anantioxidant such as ascorbic acid. As an example of an injectable oilvehicle, see Minto (1997) J. Pharmacol. Exp. Ther. 281:93-102.

Pharmaceutical formulations used to practice the invention can also bein the form of oil-in-water emulsions. The oily phase can be a vegetableoil or a mineral oil, described above, or a mixture of these. Suitableemulsifying agents include naturally-occurring gums, such as gum acaciaand gum tragacanth, naturally occurring phosphatides, such as soybeanlecithin, esters or partial esters derived from fatty acids and hexitolanhydrides, such as sorbitan mono-oleate, and condensation products ofthese partial esters with ethylene oxide, such as polyoxyethylenesorbitan mono-oleate. The emulsion can also contain sweetening agentsand flavoring agents, as in the formulation of syrups and elixirs. Suchformulations can also contain a demulcent, a preservative, or a coloringagent.

Pharmaceutical compounds used to practice the invention can also beadministered by in intranasal, intraocular and intravaginal routesincluding suppositories, insufflation, powders and aerosol formulations(for examples of steroid inhalants, see Rohatagi (1995) J. Clin.Pharmacol. 35:1187-1193; Tjwa (1995) Ann. Allergy Asthma Immunol.75:107-111). Suppositories formulations can be prepared by mixing thedrug with a suitable non-irritating excipient which is solid at ordinarytemperatures but liquid at body temperatures and will therefore melt inthe body to release the drug. Such materials are cocoa butter andpolyethylene glycols.

In practicing this invention, the pharmaceutical compounds can bedelivered by transdermally, by a topical route, formulated as applicatorsticks, solutions, suspensions, emulsions, gels, creams, ointments,pastes, jellies, paints, powders, and aerosols.

In practicing this invention, the pharmaceutical compounds can also bedelivered as microspheres for slow release in the body. For example,microspheres can be administered via intradermal injection of drug whichslowly release subcutaneously; see Rao (1995) J. Biomater Sci. Polym.Ed. 7:623-645; as biodegradable and injectable gel formulations, see,e.g., Gao (1995) Pharm. Res. 12:857-863 (1995); or, as microspheres fororal administration, see, e.g., Eyles (1997) J. Pharm. Pharmacol.49:669-674.

Slow release in the body of active ingredients used to practice thisinvention also can be administered by controlled release means or bydelivery devices that are well known to those of ordinary skill in theart; including e.g., those described in U.S. Pat. Nos. 3,845,770;3,916,899; 3,536,809; 3,598,123; and U.S. Pat. Nos. 4,008,719,5,674,533, 5,059,595, 5,591,767, 5,120,548, 5,073,543, 5,639,476,5,354,556, and 5,733,566. These formulations and dosages can be used toprovide slow or control led-release of one or more active ingredientsusing, for example, hydropropylmethyl cellulose, other polymer matrices,gels, permeable membranes, osmotic systems, multilayer coatings,microparticles, liposomes, microspheres, or a combination thereof toprovide the desired release profile in varying proportions. Suitablecontrolled-release formulations known to those of ordinary skill in theart can be readily selected for use with the active ingredients used topractice this invention. Practicing this invention also encompassessingle unit dosage forms, e.g., suitable for injection, spray and/ororal administration such as, but not limited to, tablets, capsules,gelcaps, and caplets that are adapted for controlled-release.

In practicing this invention, the pharmaceutical compounds can beparenterally administered, such as by intravenous (IV) administration oradministration into a body cavity or lumen of an organ. Theseformulations can comprise a solution of active agent dissolved in apharmaceutically acceptable carrier. Acceptable vehicles and solventsthat can be employed are water and Ringer's solution, an isotonic sodiumchloride. In addition, sterile fixed oils can be employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid can likewise be used in the preparation ofinjectables. These solutions are sterile and generally free ofundesirable matter. These formulations may be sterilized byconventional, well known sterilization techniques. The formulations maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, toxicity adjusting agents, e.g., sodium acetate, sodiumchloride, potassium chloride, calcium chloride, sodium lactate and thelike. The concentration of active agent in these formulations can varywidely, and will be selected primarily based on fluid volumes,viscosities, body weight, and the like, in accordance with theparticular mode of administration selected and the patient's needs. ForIV administration, the formulation can be a sterile injectablepreparation, such as a sterile injectable aqueous or oleaginoussuspension. This suspension can be formulated using those suitabledispersing or wetting agents and suspending agents. The sterileinjectable preparation can also be a suspension in a nontoxicparenterally-acceptable diluent or solvent, such as a solution of 1,3-butanediol. The administration can be by bolus or continuous infusion(e.g., substantially uninterrupted introduction into a blood vessel fora specified period of time).

In alternative embodiments, compounds used to practice the invention arealso formulated using cyclodextrins or cycloamyloses, e.g., to takeadvantage of the ability of cyclodextrins to form complexes withhydrophobic molecules, including inhibitory small molecules.Mechanically-interlocked molecules structures and architectures, such asrotaxanes and catenanes, can be made using compounds used to practicethis invention and cyclodextrins. Cyclodextrins used in theseembodiments can be any cyclic oligosaccharide, e.g., composed of 5 ormore α-D-glucopyranoside units linked 1→4, as in amylose, a fragment ofstarch, or, a cyclodextrins comprising glucose monomers ranging from sixto eight units in a ring, creating a cone shape α-cyclodextrin: sixmembered sugar ring molecule, or β-cyclodextrin: seven sugar ringmolecule, or γ-cyclodextrin: eight sugar ring molecule. Othercyclodextrins or cycloamyloses that can be used in formulations of thisinvention are described in, e.g., U.S. Pat. App. Pub. Nos. 20080119431(describing Per-6-guanidino-, alkylamino-cyclodextrins); 20080091006(describing nitrate ester cyclodextrin complexes); 20080058427(describing water-soluble, cyclodextrin-containing polymers with alinear polymer chain for drug delivery); 20070259931; 20070232567;20070232566; and see also U.S. Pat. No. 7,307,176 (describing a2-hydroxypropyl-beta-cyclodextrin drug inclusion complex); U.S. Pat. No.7,270,808 (describing cyclodextrin-containing polymers improve drugstability and solubility, and reduce toxicity of a small moleculetherapeutic when used in vivo); U.S. Pat. Nos. 7,262,165; 7,259,153;7,235,186; 7,157,446; 7,141,555.

The pharmaceutical compounds and formulations used to practice theinvention can be lyophilized. A stable lyophilized formulationcomprising a composition used to practice the invention can be made bylyophilizing a solution comprising a pharmaceutical used to practicethis invention and a bulking agent, e.g., mannitol, trehalose,raffinose, and sucrose or mixtures thereof. A process for preparing astable lyophilized formulation can include lyophilizing a solution about2.5 mg/mL protein, about 15 mg/mL sucrose, about 19 mg/mL NaCl, and asodium citrate buffer having a pH greater than 5.5 but less than 6.5.See, e.g., U.S. patent app. no. 20040028670.

The compositions and formulations used to practice the invention can bedelivered by the use of liposomes (see also discussion, below). By usingliposomes, particularly where the liposome surface carries ligandsspecific for target cells, or are otherwise preferentially directed to aspecific organ, one can focus the delivery of the active agent intotarget cells in vivo. See, e.g., U.S. Pat. Nos. 6,063,400; 6,007,839;Al-Muhammed (1996) J. Microencapsul. 13:293-306; Chonn (1995) Curr.Opin. Biotechnol. 6:698-708; Ostro (1989) Am. J. Hosp. Pharm.46:1576-1587.

The methods of the invention can further comprise co-administration withother drugs or pharmaceuticals, e.g., compositions for treatingconditions, infections, pathology and/or inflammation in the CNS (e.g.,brain) caused or mediated by NFkB, IL-6, NADPH oxidase (Nox2), andsuperoxide and/or hydrogen peroxide production by a NADPH oxidase totreat, prevent and/or ameliorate, e.g., schizophrenia, psychosis,delirium. e.g., post-operative delirium, drug-induced psychosis,psychotic features associated with frailty syndrome (FS), aging,depression, dementias; to treat, prevent and/or ameliorate traumatic warneurosis, post traumatic stress disorder (PTSD) or post-traumatic stresssyndrome (PTSS), Amyotrophic Lateral Sclerosis (ALS, or Lou Gehrig'sDisease), and/or Multiple Sclerosis (MS), and cognitive, learning ormemory impairments resulting therefrom, frailty syndrome (FS), aging,and related symptoms or conditions. For example, the methods of theinvention and/or compositions and formulations used to practice thisinvention can be co-formulated with and/or co-administered withantibiotics (e.g., antibacterial or bacteriostatic peptides orproteins), particularly those effective against gram negative bacteria,fluids, cytokines, immunoregulatory agents, anti-inflammatory agents,complement activating agents, such as peptides or proteins comprisingcollagen-like domains or fibrinogen-like domains (e.g., a ficolin),carbohydrate-binding domains, and the like and combinations thereof.

Therapeutically Effective Amounts

The pharmaceuticals and formulations used to practice the invention canbe administered for prophylactic and/or therapeutic treatments. Intherapeutic applications, compositions are administered to a subjectalready suffering from a condition, infection or disease in an amountsufficient to cure, alleviate, reverse or partially arrest the clinicalmanifestations of the condition, infection, pathology or disease and itscomplications (a “therapeutically effective amount”). For example, inalternative embodiments, pharmaceutical compositions and formulationsused to practice the invention are administered in an amount sufficientto treat, prevent, reverse and/or ameliorate a pathology, condition,infection or inflammation in the central nervous system (e.g., brain)caused or mediated by IL-6. NADPH oxidase enzymes, and superoxide and/orhydrogen peroxide production by a NADPH oxidase enzymes, including forexample schizophrenia, psychosis, delirium, e.g., post-operativedelirium, drug-induced psychosis, psychotic features associated withfrailty syndrome (FS), aging, frailty syndrome (FS), depression,dementias; to treat, prevent, reverse and/or ameliorate traumatic warneurosis, post traumatic stress disorder (PTSD) or post-traumatic stresssyndrome (PTSS). Amyotrophic Lateral Sclerosis (ALS, or Lou Gehrig'sDisease), and/or Multiple Sclerosis (MS), and cognitive, learning ormemory impairments resulting therefrom.

The amount of pharmaceutical composition adequate to accomplish atherapeutic effect is defined as a “therapeutically effective dose.” Thedosage schedule and amounts effective for this use, i.e., the “dosingregimen,” will depend upon a variety of factors, including the stage ofthe disease or condition, the severity of the disease or condition, thegeneral state of the patient's health, the patient's physical status,age and the like. In calculating the dosage regimen for a patient, themode of administration also is taken into consideration.

The dosage regimen also takes into consideration pharmacokineticsparameters well known in the art, i.e., the active agents' rate ofabsorption, bioavailability, metabolism, clearance, and the like (see,e.g., Hidalgo-Aragones (1996) J. Steroid Biochem. Mol. Biol. 58:611-617;Groning (1996) Pharmazie 51:337-341; Fotherby (1996) Contraception54:59-69; Johnson (1995) J. Pharm. Sci. 84:1144-1146; Rohatagi (1995)Pharmazie 50:610-613; Brophy (1983) Eur. J. Clin. Pharmacol. 24:103-108;the latest Remington's, supra).

The state of the art allows the clinician to determine the dosageregimen for each individual patient, active agent and disease orcondition treated. Guidelines provided for similar compositions used aspharmaceuticals can be used as guidance to determine the dosageregiment, i.e., dose schedule and dosage levels, administered practicingthe methods of the invention are correct and appropriate.

Single or multiple administrations of formulations can be givendepending on the dosage and frequency as required and tolerated by thepatient. The formulations should provide a sufficient quantity of activeagent to effectively treat, prevent or ameliorate a conditions, diseasesor symptoms as described herein. For example, an exemplarypharmaceutical formulation for oral administration of an inhibitorycomposition used to practice this invention can be in a daily amount ofbetween about 0.1 to 0.5 to about 20, 50, 100 or 1000 or more ug perkilogram of body weight per day. In an alternative embodiment, dosagesare from about 1 mg to about 4 mg per kg of body weight per patient perday are used. Lower dosages can be used, in contrast to administrationorally, into the blood stream, into a body cavity or into a lumen of anorgan. Substantially higher dosages can be used in topical or oraladministration or administering by powders, spray or inhalation. Actualmethods for preparing parenterally or non-parenterally administrableformulations will be known or apparent to those skilled in the art andare described in more detail in such publications as Remington's, supra.

For determining and/or optimizing the therapeutically effective amountof a composition used to practice this invention, the clinician can useany diagnostic or evaluation method or technique to determineimprovement in the patient, e.g., that administering a composition usedto practice this invention to an individual is effective to prevent,treat and/or ameliorate schizophrenia, psychosis, delirium, e.g.,post-operative delirium, drug-induced psychosis, psychotic featuresassociated with frailty syndrome (FS), aging, depression, dementias; totreat, prevent, reverse and/or ameliorate traumatic war neurosis, posttraumatic stress disorder (PTSD) or post-traumatic stress syndrome(PTSS), Amyotrophic Lateral Sclerosis (ALS, or Lou Gehrig's Disease),and/or Multiple Sclerosis (MS), and cognitive, learning or memoryimpairments resulting therefrom. In alternative embodiments, a method ofthe invention is effective if it ameliorates. e.g., improves in anydetectable or quantifiable way, or slows the progression or beginningof, or decreases in any measurable or assessable way any symptom oreffect, or reverses in any measurable or assessable way any symptom oreffect caused by schizophrenia, psychosis, delirium, e.g.,post-operative delirium, drug-induced psychosis, psychotic featuresassociated with frailty syndrome (FS), aging, depression, dementias;traumatic war neurosis, post traumatic stress disorder (PTSD) orpost-traumatic stress syndrome (PTSS), Amyotrophic Lateral Sclerosis(ALS, or Lou Gehrig's Disease), and/or Multiple Sclerosis (MS), orcognitive, learning or memory impairments resulting therefrom.

For example, schizophrenia, psychosis, delirium, e.g., post-operativedelirium, drug-induced psychosis, psychotic features associated withfrailty syndrome (FS), aging, depression, dementias; traumatic warneurosis, post traumatic stress disorder (PTSD) or post-traumatic stresssyndrome (PTSS), Amyotrophic Lateral Sclerosis (ALS, or Lou Gehrig'sDisease), and/or Multiple Sclerosis (MS), cognitive, learning or memoryimpairments resulting therefrom, and related conditions can be diagnosedand/or assessed (e.g., determining the progress, regression and/orseverity of) by the clinician using DSM-IV or DSM-IV-TR editions (e.g.,using the latest, or year 2000, edition, American PsychiatricAssociation) criteria. In alternative embodiments, methods and apparatusfor diagnosing schizophrenia, schizophrenia disorder subgroups, orpredispositions to schizophrenia disorders can be used as describede.g., in U.S. Pat. Nos. 7,338,455; 6,629,935; 5,852,489. In oneembodiment, the interhemispheric switch rate of a patient is measuredunder conditions of increasing rate of dichoptic reversal, and comparingthe switch rate with a corresponding reference switch rate to diagnosepresence or absence of schizophrenia; the interhemispheric switch ratecan be determined by measuring a rate of perceptual rivalry, e.g., bymeasuring a rate of binocular rivalry or perceptual alternations.

In alternative embodiments, to assess depression the Hamilton DepressionScale (HDS or HAMD), which is a test for measuring the severity ofdepressive symptoms in individuals, often those who have already beendiagnosed as having a depressive disorder, can be used. HDS is alsoknown as the Hamilton Rating Scale for Depression (HRSD) or the HamiltonDepression Rating Scale (HDRS). HDS is used to assess the severity ofdepressive symptoms present in both children and adults. See also U.S.Pat. No. 7,346,395, describing use of HDS to evaluate depressivesymptoms.

In alternative embodiment, compositions and methods of this inventionare used to ameliorate traumatic war neurosis (combat stress), posttraumatic stress disorder (PTSD) or post-traumatic stress syndrome(PTSS); diagnostic criteria for PTSD, per the Diagnostic and StatisticalManual of Mental Disorders IV (Text Revision) (DSM-IV-TR), can besummarized as:

-   -   A. Exposure to a traumatic event;    -   B. Persistent re-experience (e.g. flashbacks, nightmares);    -   C. Persistent avoidance of stimuli associated with the trauma;        e.g. inability to talk about things even related to the        experience; avoidance of things and discussions that trigger        flashbacks and re-experiencing symptoms. Fear of losing control;    -   D. Persistent symptoms of increased arousal, e.g. difficulty        falling or staying asleep, anger and hyper-vigilance;    -   E. Duration of symptoms more than 1 month;    -   F. Significant impairment in social, occupational, or other        important areas of functioning, e.g. problems with work and        relationships.

Criterion A (the “stressor”) can consists of two parts, both of whichmust apply for a diagnosis of PTSD. The first (A1) requires that “theperson experienced, witnessed, or was confronted with an event or eventsthat involved actual or threatened death or serious injury, or a threatto the physical integrity of self or others.” The second (A2) requiresthat “the person's response involved intense fear, helplessness, orhorror.”

Diagnosis and assessment of ALS and MS are well known in the art; forexample, in ALS, cognitive function is generally spared except incertain situations such as when ALS is associated with frontotemporaldementia, ALS also can have subtle cognitive changes of thefrontotemporal type in many patients when detailed neuropsychologicaltesting is employed. In ALS, a small percentage of patients can developfrontotemporal dementia characterized by profound personality changes;this is more common among those with a family history of dementia. Alarger proportion of ALS patients experience mild problems withword-generation, attention, or decision-making; cognitive function maybe affected as part of the disease process or could be related to poorbreathing at night (nocturnal hypoventilation).

Nanoparticles and Liposomes

The invention also provides nanoparticles and liposomal membranescomprising compounds used to practice this invention which can targetspecific molecules, including biologic molecules, such as polypeptides,including cell surface polypeptides, e.g., for targeting the inhibitorycompounds used to practice this invention to neurons in the brain, e.g.,parvalbumin (PA)-positive interneurons. Thus, in alternativeembodiments, the invention provides nanoparticles and liposomalmembranes targeting neuronal cells such as PA-positive interneurons.

In alternative embodiments, the invention provides nanoparticles andliposomal membranes comprising (in addition to comprising compounds usedto practice this invention) molecules, e.g., peptides or antibodies,that selectively target neurons in the brain, e.g., parvalbumin(PA)-positive interneurons. In one aspect, the compositions used topractice this invention are specifically designed to cross theblood-brain barrier (BBB).

The invention also provides nanocells to allow the sequential deliveryof two different therapeutic agents with different modes of action ordifferent pharmacokinetics, at least one of which comprises acomposition of this invention. A nanocell is formed by encapsulating ananocore with a first agent inside a lipid vesicle containing a secondagent; see, e.g., Sengupta, et al., U.S. Pat. Pub. No. 20050266067. Theagent in the outer lipid compartment is released first and may exert itseffect before the agent in the nanocore is released. The nanocelldelivery system may be formulated in any pharmaceutical composition fordelivery to patients. For example, one agent can be contained in theouter lipid vesicle of the nanocell, and another agent used to practicethis invention can be loaded into the nanocore. This arrangement allowsthe one agent to be released first.

The invention also provides multilayered liposomes comprising compoundsused to practice this invention, e.g., for transdermal absorption, e.g.,as described in Park, et al., U.S. Pat. Pub. No. 20070082042. Themultilayered liposomes can be prepared using a mixture of oil-phasecomponents comprising squalane, sterols, ceramides, neutral lipids oroils, fatty acids and lecithins, to about 200 to 5000 nm in particlesize, to entrap a composition of this invention.

A multilayered liposome used to practice this invention may furtherinclude an antiseptic, an antioxidant, a stabilizer, a thickener, andthe like to improve stability. Synthetic and natural antiseptics can beused, e.g., in an amount of 0.01% to 20%. Antioxidants can be used,e.g., BHT, erysorbate, tocopherol, astaxanthin, vegetable flavonoid, andderivatives thereof, or a plant-derived antioxidizing substance. Astabilizer can be used to stabilize liposome structure, e.g., polyolsand sugars. Exemplary polyols include butylene glycol, polyethyleneglycol, propylene glycol, dipropylene glycol and ethyl carbitol;examples of sugars are trehalose, sucrose, mannitol, sorbitol andchitosan, or a monosaccharides or an oligosaccharides, or a highmolecular weight starch. A thickener can be used for improving thedispersion stability of constructed liposomes in water, e.g., a naturalthickener or an acrylamide, or a synthetic polymeric thickener.Exemplary thickeners include natural polymers, such as acacia gum,xanthan gum, gellan gum, locust bean gum and starch, cellulosederivatives, such as hydroxy ethylcellulose, hydroxypropyl cellulose andcarboxymethyl cellulose, synthetic polymers, such as polyacrylic acid,poly-acrylamide or polyvinylpyrollidone and polyvinylalcohol, andcopolymers thereof or cross-linked materials.

Liposomes can be made using any method, e.g., as described in Park, etal., U.S. Pat. Pub. No. 20070042031, including method of producing aliposome by encapsulating a therapeutic product comprising providing anaqueous solution in a first reservoir; providing an organic lipidsolution in a second reservoir, wherein one of the aqueous solution andthe organic lipid solution includes a therapeutic product; mixing theaqueous solution with said organic lipid solution in a first mixingregion to produce a liposome solution, wherein the organic lipidsolution mixes with said aqueous solution so as to substantiallyinstantaneously produce a liposome encapsulating the therapeuticproduct; and immediately thereafter mixing the liposome solution with abuffer solution to produce a diluted liposome solution.

In one embodiment, liposome compositions comprising substituted ammoniumand/or polyanions are used, particularly for targeting delivery of acompound used to practice this invention to the brain, as described,e.g., in U.S. Pat. Pub. No. 20070110798.

The invention also provides nanoparticles comprising compounds used topractice this invention in the form of drug-containing nanoparticles(e.g., a secondary nanoparticle), as described, e.g., in U.S. Pat. Pub.No. 20070077286. In one embodiment, the invention provides nanoparticlescomprising a fat-soluble drug of this invention or a fat-solubilizedwater-soluble drug to act with a bivalent or trivalent metal salt.

Transport Agents for Crossing the Blood-Brain Barrier

In alternative embodiments, the invention provides pharmaceuticalcompositions and formulations, including nanoparticles and liposomalmembranes, that can cross the blood brain barrier and/or can selectivelytarget neurons in the brain, e.g., parvalbumin (PA)-positiveinterneurons. In one aspect, the compositions (including pharmaceuticalcompositions and formulations) used to practice this invention arespecifically designed to cross the blood-brain barrier (BBB). Forexample, alternative embodiments include delivering compositions used topractice this invention across the BBB include liposome-based methods,where a therapeutic agent is encapsulated within a carrier; syntheticpolymer-based methods, where particles are created using syntheticpolymers to achieve precisely-defined size characteristics; and/ordirect conjugation of a carrier to a drug, where the therapeutic agentis bound to (e.g., covalently bound to) a peptide or polypeptidecarrier, which can be synthetic or natural, e.g., as the ligand insulinfor uptake via transcytosis mediated by the endothelial insulinreceptor. Any natural or synthetic ligand (including antibodies andsmall molecules) that specifically bind to the insulin receptor,transferrin receptor, leptin receptor, lipoprotein receptor and/orinsulin-like growth factor (IGF) receptor can be used to cross the BBB.

Specific transporters for glucose or for large amino acids such astryptophan also can be used to cross the BBB. Cationized albumin or theOX26 monoclonal antibody to the transferrin receptor also can be used tocross the BBB by absorptive-mediated and receptor-mediated transcytosis,respectively. Cationized monoclonal antibodies also can be used to crossthe BBB. Antibodies that bind brain (BBB) endothelial cell receptorsresulting in endocytosis/transcytosis of the receptor and a boundligand, such as a composition (including pharmaceuticals andformulations) used to practice this invention, are also described e.g.in U.S. Pat. App. Pub. No. 20080019984.

For example, in one aspect, crossing the blood-brain barrier (BBB) canbe accomplished by incorporating BBB protein transport peptides: such asthe pentapeptide AAEAP, as described e.g. in U.S. Pat. App. Pub. No.20080213185; or polypeptides comprising at least 10% basic amino acidresidues such as arginine or lysine that have brain-localizing activityas described e.g. in U.S. Pat. App. Pub. No. 20080199436.

Ubiquinone analogs and reduced ubiquinone (ubiquinol) analogs also canbe used to cross the BBB as described e.g. in U.S. Pat. App. Pub. No.20070203080.

Another alternative embodiment encompasses an artificial low-densitylipoprotein (LDL) carrier system for the targeted delivery therapeuticagents across the BBB, e.g., using artificial LDL particles comprisingvarious lipid elements such as phosphatidyl choline,fatty-acyl-cholesterol esters, and apolipoproteins as described e.g., inU.S. Pat. App. Pub. Nos. 20080160094; 20070292413; 20070264351.Artificial low-density lipoprotein particles can facilitate transport oftherapeutic agents across the BBB by transcytosis. The BBB contains typeII scavenger receptors which bind LDL with high affinity. For example,one embodiment comprises use of an artificial LDL particle comprising anouter phospholipid monolayer and a solid lipid core, where the outerphospholipid monolayer comprises at least one apolipoprotein and thesolid lipid core contains at least one therapeutic agent.

Synthetic polymers such as a poly(butyl cyanoacrylate) or apolyacrylamide covered with a polysorbate (e.g., POLYSORBATE 80) can beused because these particles are sufficiently hydrophilic to bewater-soluble, yet are able to maintain their structural form for longperiods, which protects the therapeutic agent from uptake into the liverand kidney where it is subject to natural detoxification process.

Another alternative embodiment encompasses use of synthetic poly(butylcyanoacrylate) particles to which ApoE molecules are covalently bound.The surface of the particles are further modified by surfactants orcovalent attachment of hydrophilic polymers, see e.g., U.S. Pat. No.6,288,040.

Devices for Delivering Therapeutic Agents Directly into the Brain

In alternative embodiments, pharmaceutical compositions andformulations, including nanoparticles and liposomes, used to practicethis invention are delivered directly into the brain, e.g., by variousdevices known in the art. For example, U.S. Pat. App. Pub. No.20080140056, describes a rostrally advancing catheter in the intrathecalspace for direct brain delivery of pharmaceuticals and formulations.Implantable infusion devices can also be used; e.g., a catheter todeliver fluid from the infusion device to the brain can be tunneledsubcutaneously from the abdomen to the patient's skull, where thecatheter can gain access to the individual's brain via a drilled hole.Alternatively, a catheter may be implanted such that it delivers theagent intrathecally within the patient's spinal canal. Flexible guidecatheters having a distal end for introduction beneath the skull of apatient and a proximal end remaining external of the patient also can beused, e.g., see U.S. Pat. App. Pub. No. 20060129126.

In alternative embodiments, pharmaceutical compositions and formulationsused to practice this invention are delivered via direct implantation ofcells into a brain, for example, using any cell implantation cannula,syringe and the like, as described e.g., in U.S. Pat. App. Pub. No.20080132878; or elongate medical insertion devices as described e.g., inU.S. Pat. No. 7,343,205; or a surgical cannula as described e.g., inU.S. Pat. No. 4,899,729. Implantation cannulas, syringes and the likealso can be used for direct injection of liquids, e.g., as fluidsuspensions.

In alternative embodiments, pharmaceutical compositions and formulationsused to practice this invention are delivered with tracers that aredetectable, for example, by magnetic resonance imaging (MRI) and/or byX-ray computed tomography (CT); the tracers can be co-infused with thetherapeutic agent and used to monitor the distribution of thetherapeutic agent as it moves through the target tissue, as describede.g., in U.S. Pat. No. 7,371,225.

Drug Discovery

The methods and compositions of the invention can be used in drugdiscovery. The methods and compositions of the invention can be used fortarget validation; and, in some applications, can provide aphysiologically accurate and less expensive approach to screen potentialdrugs to treat schizophrenia, a psychosis, a dementia, delirium,depression, traumatic war neurosis, post traumatic stress disorder(PTSD) or post-traumatic stress syndrome (PTSS), Amyotrophic LateralSclerosis (ALS, or Lou Gehrig's Disease), and/or Multiple Sclerosis(MS), and the like, and/or to developing brain-targeted NADPH oxidaseinhibitors. For example, in one aspect, the methods and compositions ofthe invention are used to validate the efficacy of a treatment and/or adrug for any disease, condition, genetic phenotype (e.g., a syndrome),toxic effect (e.g., poisoning), infection and/or trauma, involving aninflammation or an inflammatory component in the CNS (e.g., brain)caused or mediated by NFkB, IL-6, NADPH oxidase enzymes, and superoxideand/or hydrogen peroxide production by a NADPH oxidase; includingMultiple Sclerosis (MS), Progressive Multifocal Leuko-encephalopathy,HIV encephalitis, including any neurodegenerative disease with a CNSinflammatory component (including a CNS inflammatory component caused bya treatment, such as a drug)—such as Alzheimer's disease, Lewy BodyDisease, Parkinson's Disease, Huntington's Disease, Multi-infarctdementia, senile dementia or Frontotemporal Dementia, AmyotrophicLateral Sclerosis (ALS, or Lou Gehrig's Disease), and/or MultipleSclerosis (MS), and related diseases, infections and/or geneticconditions.

Kits and Instructions

The invention provides kits comprising compositions and methods of theinvention, including instructions for use thereof. As such, kits, cells,vectors and the like can also be provided.

The invention provides kits comprising a composition that inhibits NFkB,IL-6, NADPH oxidase enzymes, and/or superoxide and/or hydrogen peroxideproduction by any member of the NADPH oxidase enzyme family (e.g., Nox1,Nox2, Nox3, Nox4 or Nox5), and in an alternative embodiment, the kitcomprises instructions for using a method of the invention. Alsoprovided are kits having instructions for ameliorating, preventing orreversing schizophrenia, psychosis, delirium, e.g., post-operativedelirium, drug-induced psychosis, psychotic features associated withfrailty syndrome (FS), aging, depression, dementias; or forameliorating, preventing or reversing traumatic war neurosis, posttraumatic stress disorder (PTSD) or post-traumatic stress syndrome(PTSS), Amyotrophic Lateral Sclerosis (ALS, or Lou Gehrig's Disease),and/or Multiple Sclerosis (MS), or cognitive, learning or memoryimpairments resulting therefrom, by practicing the methods of theinvention.

The invention will be further described with reference to the followingexamples; however, it is to be understood that the invention is notlimited to such examples.

EXAMPLES Example 1: Brain NADPH-Oxidase Mediates Ketamine Effects onParvalbumin-Expressing Fast-Spiking Interneurons

This example demonstrates that the compositions and methods of theinvention are effective in the amelioration of conditions, pathologies,inflammation and/or infections in the central nervous system. e.g.,brain, caused or mediated by NFkB, IL-6, NADPH oxidase, and superoxideand/or hydrogen peroxide production by a NADPH oxidase, including forexample the amelioration or prevention of schizophrenia, psychosis,delirium, e.g., post-operative delirium, drug-induced psychosis,psychotic features associated with frailty syndrome (FS), aging,depression, dementias, traumatic war neurosis, post traumatic stressdisorder (PTSD) or post-traumatic stress syndrome (PTSS). Thecompositions and methods of this invention can be used to inhibit ordecrease the amount of NFkB, IL-6, NADPH oxidase, and/or superoxideand/or hydrogen peroxide production by inhibiting or decreasing theactivity of the enzyme NADPH oxidase and/or IL-6 and/or IL-6 receptor.

This invention demonstrates that NADPH oxidase is responsible fordysfunction of the parvalbumin (PV)-positive interneurons, and thatinhibiting NADPH oxidase rescues these same neurons. This inventiondemonstrates that interleukin-6 (IL-6) is responsible for the inductionand activation of NADPH oxidase, and that inhibition of IL-6 activityprevents the deleterious effects of Nox activation of PV-positiveinterneurons; thus demonstrating that the compositions and methods ofthe invention can be used to treat, ameliorate or prevent schizophrenia,psychosis, post-operative delirium, drug-induced psychosis, psychoticfeatures associated with frailty syndrome (FS), aging, depression,dementias, traumatic war neurosis, post traumatic stress disorder (PTSD)or post-traumatic stress syndrome (PTSS).

The inventors found that prolonged use of ketamine induces neuronalNADPH-oxidase, which in turn has deleterious effects on PV-interneurons,and that embodiments of the compositions and methods of this inventioncan decrease or reverse this neuronal NADPH-oxidase increase andameliorate these deleterious effects, such as schizophrenia, psychosis,delirium, e.g., post-operative delirium, drug-induced psychosis,psychotic features associated with frailty syndrome (FS), aging,depression, dementias, traumatic war neurosis, post traumatic stressdisorder (PTSD) or post-traumatic stress syndrome (PTSS).

The compositions and methods of this invention can be used to ameliorateor prevent the negative (deleterious) effects of ketamineadministration, including chronic or improper ketamine administration,or abuse of ketamine, or other NMDA-receptor antagonists; these negativeeffects can include a syndrome indistinguishable from schizophrenia.Ketamine's acute pro-psychotic effects occur through dis-inhibition ofbrain circuitry caused by diminished firing of cortical fast-spikinginhibitory interneurons. However, after prolonged use, brain activitydecreases and a loss of expression of the GABA-producing enzymeglutamate decarboxylase 67 (GAD67) (indicating loss of GABAergicphenotype), and of parvalbumin develops in these interneurons through anunknown mechanism. The inventors found that prolonged use of ketamineinduces neuronal NADPH-oxidase; it was found that prolonged ketamineexposure in mice induces a persistent increase in brain superoxide dueto induction of neuronal NADPH-oxidase.

Decreasing superoxide and/or hydrogen peroxide production with apocyninor inhibiting its actions with a carboxyfullerene-based SOD-mimeticprevented the deleterious effects of ketamine on inhibitory interneuronsin mouse prefrontal cortex. This invention identifies NADPH-oxidase as anovel avenue in the treatment of ketamine-induced psychosis andschizophrenia, and provides compositions and methods for ameliorating orpreventing ketamine-induced psychosis and schizophrenia (and associatedpsychosis, delirium, e.g., post-operative delirium, drug-inducedpsychosis, psychotic features associated with frailty syndrome (FS),aging, depression, dementias, traumatic war neurosis, post traumaticstress disorder (PTSD) or post-traumatic stress syndrome (PTSS).

Exposure to the NMDA-receptor (NMDA-R) antagonists phencyclidine andketamine, while reproducing schizophrenia symptoms in healthy humanvolunteers (see reference 1, below), induces an initial dis-inhibitionof excitatory transmission in the PFC of rodents and non-human primates(see reference 2, below), which, after prolonged exposure, is followedby a depression in brain activity (see reference 3, below), and by lossof the GABAergic phenotype of fast-spiking parvalbumin-positive (PV)inhibitory interneurons. This loss of GABAergic phenotype includes areduced expression of GAD67, the main isoform producing GABA, as well asthat of the calcium binding protein parvalbumin and the GABA transporter1 (see references 4 and 5, below), similarly to what was described inthe dorsolateral prefrontal cortex (PFC) of postmortemschizophrenic-brain samples (see reference 6, below, for review).

PV-interneurons are involved in the generation of gamma oscillationsresponsible for temporal-encoding and storage/recall of informationrequired for working memory (see reference 7, below). These interneuronsreceive the highest glutamatergic input amongst all GABAergic neurons incortex (see reference 8, below), and their basal synaptic activation iscontrolled by calcium entry through NMDA-Rs (see reference 9, below).The subunit composition of these glutamate receptors in PV-interneuronsdiffers from those present in neighboring pyramidal neurons (seereference 10, below), and are highly sensitive to NMDA-R antagonistssuch as ketamine (see reference 11, below).

The mechanism(s) by which the initial dis-inhibition of excitatorytransmission created by NMDA-Rs leads to the delayed, or compensatoryhypo-function of the system and to the decreased firing ofPV-interneurons, are not known.

Therefore, the inventors studied whether this initial dis-inhibition iscritical to the subsequent loss of GABAergic phenotype by studying theeffects of the pan-GABA_((A)) agonist muscimol in reversingketamine-mediated effects on cultured PV-interneurons, a systempreviously shown to respond to ketamine treatment with reductions inparvalbumin and GAD67 immunoreactivity (see reference 10, below).Increasing GABA_((A)) mediated inhibition prevented the decrease inparvalbumin (FIG. 1A-1E) and GAD67 (FIG. 5A-5B) in PV-interneurons,confirming that increased excitability is a key initial event afterketamine exposure (see reference 2, below).

Rapid increases in reactive oxygen-species production have been shownupon exposure to NMDA-R antagonists in vitro (see reference 12, below),and in vivo (see reference 13, below), but the processes initiating thisincrease are not clear (see reference 14, below). Interestingly, one ofthe most consistent findings in microarray analyses of schizophrenicbrain tissue, as well as in animal models of the disease, is an increasein oxidative- and inflammatory-related gene transcripts; see (seereference 14, below) for review. Diminished antioxidant capacity inplasma and CSF of schizophrenic patients has also been shown (seereferences 16 and 17, below), supporting the hypothesis of increasedoxidative-stress in the disease.

Expression of the superoxide-producing enzyme NADPH-oxidase (Nox) inhippocampus has been demonstrated; see e.g., see reference 18, below.

The inventors showed that dis-inhibition of neurotransmission by NMDA-Rantagonists leads to an increase in Nox-activity. Superoxide and/orhydrogen peroxide production in live cells has been successfullydetected by dihydroethidium (DHE) oxidation (see references 19 to 21,below). Therefore, following the oxidation product of DHE by confocalmicroscopy, levels of superoxide and/or hydrogen peroxide productionwere analyzed after prolonged exposure to low concentrations of ketaminein the culture system. We observed a significant increase in neuronalsuperoxide and/or hydrogen peroxide production after 24 h exposure to0.5 μM ketamine, which was prevented by the GABA_((A)) agonist muscimol(FIGS. 1A, and B left graph) and was accompanied by the reduction ofparvalbumin immunoreactivity (FIGS. 1A, and B right graph). The increasein DHE oxidation in response to ketamine was not restricted to thePV-interneuronal population, demonstrating that activation of theenzyme(s) producing superoxide occurs throughout cortical neurons.

It was next determined whether the increase in superoxide and/orhydrogen peroxide production was involved in the loss of GABAergicphenotype of PV-interneurons in the culture system. It was found thatketamine effects were prevented by co-treatment with acarboxyfullerene-based SOD-mimetic (C₃) (FIGS. 2A and B) (21).

Nox2 and Nox4 are the main Nox core-subunits expressed in forebrain(22). Nox2, is the main isoform expressed in professional phagocytes andrequires the presence of the membrane protein p22^(phox), as well as ofa series of cytosolic proteins involved in the priming and activation ofthe enzyme, i.e. p47^(phox), p67^(phox), p40^(phox) and Rac1. Activationof the Nox2-complex occurs upon bacterial infection and inflammatoryprocesses. Nox4 is also dependent on p22^(phox) for activity, but seemsto be a constitutive enzyme not requiring activation by the cytosoliccomplex (22).

To test if Nox activity was involved in ketamine-mediated superoxideincrease the inventors used the Nox inhibitor apocynin, which acts bypreventing binding of p47^(phox) to p22^(phox) required for Noxactivation (23). When cultures were exposed to ketamine in the presenceof apocynin (Apo 0.5 mM) superoxide and/or hydrogen peroxide productionwas significantly reduced (FIG. 2A), and the loss of parvalbumin andGAD67 immunoreactivity in PV-interneurons was prevented (FIG. 2B).Furthermore, ketamine induced the neuronal expression of Nox2 (seeFigure S2).

To determine whether Nox-dependent superoxide was also important inketamine effects in vivo, a sub-chronic regimen was used that consistedof intraperitoneal (IP) injections of ketamine at 30 mg/kg applied ontwo consecutive days to male C57BL/6 mice, followed by brain dissection18 hours later. The acute effects of ketamine, such as behavioraleffects due to disinhibition (2), are not detected in this regimen.However, this treatment permits the analysis of events that follow theinitial dis-inhibition of the circuitry. A significant increase wasobserved in the expression of Nox2 and p22^(phox), but not Nox4 (FIG.3A) in membrane preparations of cortical tissue after ketaminetreatment. This increase in protein levels was accompanied by anincrease in Nox activity in synaptosomes isolated from cortical tissueof ketamine treated animals (FIG. 3B), demonstrating a synapticlocalization of the active enzyme. The increased oxidase activity insynaptosomes was inhibited in vitro by apocynin (FIG. 3B), confirmingthat the main oxidase isoform induced by ketamine in brain is Nox2.Metabolic activities of synaptosomal mitochondria were not affected bythe treatment, indicating that this potential source of ROS is notinvolved in ketamine effects in vivo (Figure S3).

To assess the role of Nox activation and superoxide and/or hydrogenperoxide production in the effects of ketamine on PV-interneurons, theseinterneurons were characterized in mouse PFC and analyzed the effects ofthe two-day ketamine regimen on parvalbumin and GAD67 immunoreactivity.We observed a significant reduction in immunoreactivity for bothproteins in PFC after ketamine treatment (FIG. 4A). Moreover, thistreatment produced a widespread increase in oxidized DHE (FIGS. 4B andC), indicating increased superoxide and/or hydrogen peroxide production,which was prevented when animals were pretreated with the Nox inhibitorapocynin (5 mg/kg/day for 1 week in drinking water), or with theSOD-mimetic C₃ for one month; 1.0 mg/kg/day, ALZET™ (Cupertino, Calif.)mini-pumps. More importantly, both treatments completely prevented theketamine-mediated loss of parvalbumin immunoreactivity inPV-interneurons (FIG. 4D).

Although the PFC seems to be more susceptible to the effects of NMDAreceptor antagonists (4, 5), structural and functional deficits inhippocampus, visual and auditory regions have been shown to contributeto schizophrenia (24, 25). Substantial increases in oxidized DHE wasobserved in several brain regions besides the PFC, such as CA3 in thehippocampus and the reticular nucleus of the thalamus (see FIG. 4A-4G)demonstrating that increased Nox activity occurs throughout the brainupon drug exposure.

Regulatory redox sites have been found in many proteins that areinvolved in glutamatergic neurotransmission including the excitatoryamino acid transporter EAAT1 (26), serine-racemase (27), and the NMDAreceptor itself which is tightly regulated by oxidation-reductionreactions through its redox-sensitive site (28, 29). Redox agents,including glutathione, induce a highly reversible current potentiationin receptors composed of NR1:NR2A by acting on a specific redox site inNR2A, and the oxidation status of this site affects the physiologicalregulation of the receptor.

The higher ratio of NR2A-containing NMDA-Rs in PV-interneurons (10)should make these cells highly sensitive to changes in oxidativeconditions. It appears then that a prolonged inactivation of NMDA-Rs inPV-interneurons either by blockade with the antagonists, or, morephysiologically, by Nox-dependent oxidation leads to a“misinterpretation” of the lack of signal through NMDA-Rs as a decreasedglutamatergic transmission. This, in turn, would be the signal theinitiates the processes resulting in reduced expression of GABAergicmarkers and loss of inhibitory capacity in PV-interneurons, finallyleading to a chronically decreased inhibitory tone in cortex.

In summary, this invention demonstrated that the diminished firing ofPV-interneurons caused either by blockade of NMDA receptors, or bydevelopmental derangements as for schizophrenia, produces the initialincreased excitability in brain (2). This, in turn, activates andinduces Nox, which through oxidation of synaptic proteins leads todiminished neurotransmission. This sequence may function as a normalshut-down mechanism in transient situations of increased excitatorytransmission, as was recently suggested for the inactivation ofserine-racemase (27).

The inventors demonstrated that NADPH-oxidase (Nox) and superoxidedismutase (SOD) are contributors to oxidative mechanisms in thepsychotomimetic effects of NMDA-R antagonists and in schizophrenia andother processes involving increased brain oxidative-stress, such as CNSinflammation. Accordingly, the invention provides compositions andmethods for manipulating the activation/induction mechanism of brainNADPH-oxidase (Nox), and mimicking the activity of superoxide dismutase(SOD); thus, the invention presents completely new avenues for thetreatment of schizophrenia, psychosis, delirium, drug-induced psychosis,psychotic features associated with frailty syndrome (FS), aging,depression and/or dementias, traumatic war neurosis, post traumaticstress disorder (PTSD) and/or post-traumatic stress syndrome (PTSS), bymanipulating NADPH-oxidase (Nox) and/or superoxide dismutase (SOD)expression and activity.

FIG. 1. Ketamine exposure in primary neuronal cultures increasessuperoxide and/or hydrogen peroxide production and induces the loss ofparvalbumin immunoreactivity. Neuronal cultures were treated withketamine (0.5 μM) for 24 h as described (10), and DHE (1 μg/ml) wasadded to the cultures during the last hour of incubation. FIG. 1A, FIG.1B, and FIG. 1C: Confocal images of representative fields depicting aPV-interneuron and surrounding neurons treated in the absence ofketamine (control) (FIG. 1A), the presence of ketamine (FIG. 1B), andco-exposure to ketamine and muscimol (FIG. 1C). FIG. 1D and FIG. 1E:Quantification results for DHE (FIG. 1D), and PV (FIG. 1E) fluorescence.Co-exposure to muscimol (10 μM) prevented the increase in oxidized DHEand loss of PV immunoreactivity (right bars in D and E). (*=significantwhen compared to control at P<0.001 by analysis of variance (ANOVA)followed by Tukey's test, n=5 experiments per condition).

FIG. 2A-2B. Removal of superoxide or inhibition of Nox activationprevents superoxide increase and reduction of parvalbumin and GAD67 inPV-interneurons in culture. Cultures were treated with ketamine as inFIG. 1A-1E in the absence or presence of the carboxyfullerene-basedSOD-mimetic C₃ (20 μM) or the Nox inhibitor apocynin (0.5 mM).Quantification results for oxidized DHE fluorescence (FIG. 2A), and forparvalbumin and GAD67 fluorescence in PV-interneurons (FIG. 2B)(*=significant when compared to control at P<0.05 by ANOVA followed byTukey's test. n=4 experiments per condition).

FIGS. 3A-3B. In vivo ketamine treatment increases Nox and p22^(phox)protein expression in brain membranes, and increases the levels ofapocynin-inhibitable Nox activity in synaptosomes. Mice were treatedwith ketamine (30 mg/kg) on two consecutive days followed by 18 hwithout drug. FIG. 3A: Membrane fractions were analyzed for theexpression of the indicated proteins by Western blots (insert). Bargraphs represent the quantification of Western blots normalized foractin content. (*=significant compared to saline at P<0.001 by ANOVAfollowed by Tukey's test. n=4 animals/condition). FIG. 3B: Increased Noxactivity was observed in synaptosomal preparations from ketamine treatedanimals. This activity was inhibited by apocynin. Values ofNADPH-induced oxygen consumption (nmol O₂/mg protein/min) were:4.67±0.98, control; 7.9±1.8, ketamine (n=4 animals/condition).

FIGS. 4A-4G. Pretreatment of animals with the Nox inhibitor apocynin, orwith the SOD-mimetic (C₃) reduces superoxide and/or hydrogen peroxideproduction and prevents the loss of parvalbumin immunoreactivity inducedby ketamine in mouse prefrontal cortex. Animals were treated withketamine (30 mg/kg. ip) as in FIG. 3A-3B. Coronal sections comprisingthe prelimbic and infralimbic regions were analyzed. FIG. 4A;parvalbumin and GAD67 expression in PV-interneurons, graph bar of FIG.4C represents the quantification of parvalbumin and GAD67 meanfluorescence/cell for the region normalized by the means of salinetreated animals. FIG. 4B and FIG. 4C: Animals were treated with apocyninin the drinking water for 1 week (5 mg/kg/day), or during one month withthe SOD-mimetic C₃ delivered by mini-pumps (1 mg/kg/day) before ketaminetreatment. DHE was applied 30 min after the last ketamine injection.Coronal sections were quantified for parvalbumin and oxidized DHEfluorescence. n=6 animals per condition. (*,#=significance with respectto saline at the indicated P values by ANOVA followed by Tukey's test).

FIGS. 5A-5B. Increasing GABA_((A))-mediated inhibition prevents thedecrease in GAD67 expression in parvalbumin-positive (PV)-interneuronsafter ketamine treatment in primary neuronal cultures. Cultures weretreated with ketamine in the absence or presence of muscimol as in FIGS.1A-1E, above, and GAD67 immunofluorescence in PV-interneurons wasanalyzed as described in the methods section. FIG. 5A illustratesconfocal images; and FIG. 5B is a bar graph schematicallyillustrating-summarizing the data from this study; *=significance withrespect to control conditions at P=<0.001 by ANOVA followed by Tukey'stest. n=5 experiments per condition.

FIGS. 6A-6B. Ketamine treatment increases Nox2 expression in primaryneuronal cultures. FIG. 6A: Confocal image showing the increase in Nox2immunoreactivity after 24 h of treatment with ketamine (0.5 μM) inprimary cultured neurons (fluorescence quantification values: control:100+/−8%; Ketamine: 170+/−15%. Statistically significant at P<0.001 byANOVA followed by Tukey's multiple comparisons post-hoc test). FIG. 6B:Inset shows Western blots prepared form cultures treated as in FIG. 6A,showing increase in Nox2 protein level, with bar graph schematicallyillustrating-summarizing the data from this study. Cultured cells wereextracted with RIPA-buffer and Western blots were run using 50 μg ofprotein per lane. Nox2 and actin immuno-reactivities were detected asdescribed in methods, and quantified by densitometry. The Nox2/actinratios were calculated and expressed as percent of control.*=significant when compared to control conditions by ANOVA followed byTukey's test. n=3 experiments.

FIGS. 7A-7B. Ketamine effects on synaptosomal O₂ consumption by Nox(s)and mitochondria. FIG. 7A: Oxygen consumption by synaptosomal Nox(s)from cortex of saline or ketamine injected mice at 37° C. was induced bythe addition of 5 mM NADPH to samples containing 2-5 mg synaptosomalprotein. The inset in FIG. 7A shows the apocynin dependent inhibition ofNox activity. Respiratory function of synaptosomal mitochondria in thesame preparations was then evaluated by the subsequent addition ofNAD+-linked substrates (10 mM malate+10 mM pyruvate) followed by theaddition of 4 μg/ml of the F₀F₁-ATPase inhibitor oligomycin to attainState 4 respiration, and the maximal mitochondria respiration wasinitiated by the addition of 0.5 μM of the protonophore uncouplingagent, CCCP. FIG. 7B: Ketamine treatment did not affect synaptosomalmitochondria. Quantifications of OXYGRAPH™ traces similar to those shownin FIG. 7A from saline- or ketamine-injected (n=4) mice were carriedout. Data are mean±SEM.

FIG. 8. Ketamine-Mediated Decrease in Parvalbumin and GAD67Immunoreactivity in PV-Interneurons of the PFC is Prevented by ApocyninTreatment

Confocal images of parvalbumin and GAD67 stained sections of theprefrontal region depicting the decrease in immunoreactivity induced bythe two-day ketamine treatment. These decreases were prevented whenanimals were treated with apocynin in the drinking water as in FIG.4A-4G. Images were obtained with a 10× water-immersion objective.Fluorescence intensity per cell was analyzed as described in the Methodssection, below. Bar graph represents means+/−SEM values expressed as %of control (saline) conditions. *,#=significant when compared to controlconditions (saline treated animals at indicated P values by ANOVAfollowed by Tukey's test. n=5 animals per condition. Data are mean±SEM.

Example 1—Material and Methods

Animals and Treatments.

Maintenance of mice and in vivo administration of ketamine, apocynin andthe carboxyfullerene-based SOD-mimetic C₃. Male C57BL6 mice wereobtained from Jackson Labs, Bar Harbor, Me., at 8-12 weeks and housed inour facility until 15 weeks when they were used for experiments.Ketamine (30 mg/kg) was applied intraperitoneally (IP) on twoconsecutive days at around 4 pm. DHE was applied 30 min after the lastketamine injection as described (19, 20). Briefly, two serial i.p.injections of freshly prepared dihydroethidium (27 mg/kg) are given at30 minute intervals. Eighteen hours later, mice are anesthetized withinhaled halothane, and perfused intracardially with cold saline followedby 4% paraformaldehyde in PBS. The Nox inhibitor, apocynin (5 mg/kg/day)was given in the drinking water for a total of seven days, with anassumed intake of 13 ml H₂O/mouse/day, and ketamine was applied on thelast two days. The SOD mimetic C₃ was given through ALZET™ minipumps at(1 mg/kg/day) for 30 days before ketamine injections. All animal studieswere approved by the Animal Care Program at the University ofCalifornia, San Diego, and are in accordance the PHS Guide for the Careand Use of Laboratory Animals. USDA Regulations, and the AVMA Panel onEuthanasia.

Neuronal Cultures.

Neuronal cultures used for confocal imaging and biochemistry wereprepared from fetal (E14-15) Swiss Webster mice as previously described(10).

Synaptosomal Preparations.

For each preparation, pooling of two forebrains was found to providesufficient protein (10-15 mg) to run one OXYGRAPH™ measurement (seebelow) and Western blots. Mice were euthanized by exposure to a lethaldose of inhaled halothane, followed by cervical dislocation. Brains wererapidly removed, cortices dissected and homogenized in 10 volumes ofice-cold isolation buffer (0.32M sucrose, 1 mM EDTA, 10 mM Tris-HClbuffer, pH 7.4, 10 mM glucose). The homogenate was centrifuged at 3100rpm for 3 min at 4° C., and the supernatant collected, and the pelletwas re-homogenized in half the volume of isolation buffer andcentrifuged again. Synaptosomal were then isolated as described (seereference 30, below) modified as follows, the pooled supernatants weremixed with PERCOLL™ (Sigma. St. Louis, Mo.) to a final concentration of15%, and then layered onto a step gradient of 23% and 40% PERCOLL™, andcentrifuged at 16,000 rpm for 5 minutes at 4° C. The uppermost band wasextracted, rinsed in isolation buffer, centrifuged and resuspended insynaptosomal buffer (120 mM NaCl, 4.7 mM KCl, 2.2 mM CaCl₂, 1.2 mMMgCl₂, 25 mM HEPES, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 10 mM glucose).

Nox Activity.

NADPH-dependent oxygen consumption (oximetry) was used for determiningNox activity in synaptosomal preparations. Approximately (˜) 5 mgsynaptosomal protein was incubated for 10 minutes at 37° C. with 200 μMdigitonin and different apocynin concentrations (0-200 μM) before theactivation of NOX was triggered by the addition of 5 mM NADPH. Viabilityof synaptosomal mitochondria in the presence of digitonin and apocyninwas assessed by respiration upon the addition of 10 mM malate and 10 mMpyruvate. F0F1-ATPase inhibitor oligomycin (4 μg/mL) halted oxygenconsumption and the maximal mitochondria uncoupling was established bythe addition of 0.5 μM CCCP as an indication of ‘healthy mitochondria’.O₂ utilization was measured using an oxygen Clark-type electrode,OXYGRAPH™ (Hansatech, UK) with OXYGRAPH™ software.

Analysis of Nox Proteins by Western Blot.

Preparation of samples and subcellular fractionation is carried out asdescribed above. For Western blotting, samples were separated bySDS-PAGE on 10-12% acrylamide gels, proteins transferred to anitrocellulose membrane, and processed for immunodetection as described(see reference 31, below) using mouse monoclonal antibodies against Nox2(54.1; 1:1000) and p22^(phox) (44.1; 1:1000) kind gift of Dr. Quinn (seereference 32, below) and, Nox4 polyclonal (1:1500) kind gift from Dr.Goldstein (see reference 33, below), and anti-Actin (1:30000; Chemicon)and incubating at 4° C. overnight. After incubation withhost-appropriate secondary Abs HRP-conjugated, specific antigens werevisualized using chemiluminescence (SUPERSIGNAL PICO™, Pierce Chemical,Rockford, Ill.). Protein content was quantified by densitometricanalysis and normalized by the actin content in the same sample. Valueswere then expressed as % of control (saline) conditions.

Imnmunocytochemistry.

Fixation of neurons in culture was performed as described (10). Fordouble immunostaining, the coverslips were incubated in 2% normal goatserum containing the following primary antibodies (Abs): mAb againstGAD67 (1:1000, Chemicon), Nox2 or Nox4 (1:200. Kindly provided by DrQuinn), a rabbit polyclonal Ab against Parvalbumin (1:3000, Swant,Bellinzona, Switzerland), or p220^(phox) (1:300. Santa Cruz), andincubated for 2 h at 37° C. Specific binding was detected by incubationfor 45 min at room temperature with a 1:1000 dilution of secondary Absconjugated to ALEXAFLUOR™ dyes (568: red, 488: green, Molecular Probes).

Immunohistochemistry:

Brains were frontally sliced in a vibratome into 50 μm coronal sectionsencompassing the prefrontal cortex region (from Bregma 2.0 to 1.3).Sequential slices were processed for floating-section doubleimmunohistochemistry for the detection of parvalbumin and GAD67. Antigenretrieval was performed by incubation of the slices in 1% sodiumborohydride for 15 min as described (see reference 34, below), followedby washing in PBS and incubation in 10% normal goat serum in PBS for 16h at 4° C. Primary antibodies (Calbindin: 1:5000: Calretinin: 1:2000;Parvalbumin: 1:3000, all rabbit polyclonals from Swant, Bellinzona,Switzerland. GAD67: 1:1000, from Chemicon-Millipore, Temecula, Calif.)were diluted in 2% normal goat serum in PBS and applied to the slicesfor 18 h at 4° C., after which slices were washed in PBS and incubatedin a 1:1000 dilution of ALEXAFLUOR™ (Molecular Probes, Invitrogen,Carlsbad, Calif.) conjugated goat anti-rabbit (568) or goat anti-mouse(488) antibodies for 1 hr at room temperature (rt). Slices were washedin PBS and mounted sequentially in glass slides using VECTASHIELD™(Vector Laboratories, Burlingame, Calif.), covered with a coverslip andallowed to dry for at least 24 h before confocal imaging.

Confocal Microscopy and Image Analysis:

Mounted slices or coverslips were evaluated for fluorescence undersettings for 568 and 488 emissions on a LSM510 META™ multiphoton laserconfocal microscope (Karl Zeiss, Inc.) using a 10×-PLANAPO™ objective(for slices) or a 40× water immersion objective (for coverslips).Ethidium fluorescence, the DHE oxidation product, was obtained using Exλ 543 nm, Em λ>590 nm. For slice imaging, each slice was imaged acrossthe prelimbic and infralimbic regions between Bregmas 1.3 and 2.0 (threeimages per slice). Six slices were analyzed per animal. For each slice az-stack of 8 images was obtained (corresponding to 1.4 μm on the z-axis)for a total of 144 images per animal. All PV-neurons in the images wereanalyzed for their parvalbumin and GAD67 content.

Image analysis of the neuronal population in primary cultures wasessentially as described (10). Briefly, coverslips are scanned to obtain200-400 neurons (approx. 26-30 images captured per coverslip percondition using a 40× water immersion objective). Each image analyzedconsists of a stack of 16 0.2 μm Z-stage images taken from the base ofthe neurons and across 3.2 μm depth. When analyzing PV-interneurons inparticular, the coverslips are scanned to obtain images as before butfor all the PV-interneurons in the coverslip.

The settings of the confocal microscope were maintained constant foreach series of experiments so that the resulting images could beanalyzed by densitometry and the treatment-dependent changes influorescence compared and expressed as % of untreated (saline)conditions. Images were then analyzed for their somatic median green andred fluorescence content using METAMORPH™ (Molecular Devices, Sunnyvale,Calif.). The median fluorescence/cell was then averaged across allimaged slices of the same animal (or experiment in the case of primarycultures in coverslips), and the mean fluorescence intensity/cell/animalwas then expressed as percent of control (saline) conditions.

Statistical Analysis.

All values obtained per experiment were analyzed by one-way analysis ofvariance (ANOVA) followed by Tukey's post-hoc test with alpha 0.05 usingSIGMASTAT™ software (Aspire Software International, Ashburn, Va.).

Example 1 Cited References

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Example 2: Compositions and Methods of the Invention are Effective inthe Amelioration of Pathology in the Brain Caused or Mediated by IL-6,NADPH Oxidase and SOD Enzymes

This example demonstrates that the compositions and methods of theinvention are effective in the amelioration of pathology in the braincaused or mediated by IL-6, IL-6-R, NADPH oxidase, and superoxide and/orhydrogen peroxide production by a NADPH oxidase, including for exampleschizophrenia, psychosis, delirium, e.g., post-operative delirium,drug-induced psychosis, psychotic features associated with frailtysyndrome (FS), aging, depression, dementias, traumatic war neurosis,post traumatic stress disorder (PTSD) or post-traumatic stress syndrome(PTSS).

Ketamine exposure induced a pronounced increase in DHE oxidation both invivo (in the prefrontal cortex, PFC) and in cultures, as illustrated bythe data presented schematically in FIG. 9. Male C57BL/6J were treatedwith ketamine; 30 mg/kg i.p on two consecutive days, and sacrificed 18 hafter the last ketamine injection. Dihydroethidium (DHE) was applied ½hour after the last ketamine injection. DIV 21 cortical neuronalcultures were treated with ketamine (0.5 μM) for 24 h, and exposed toDHE 1 μg/ml for the last hour. PFC coronal sections and primary culturedneurons were analyzed for DHE fluorescence and parvalbuminimmunoreactivity as described herein. In both cases the secondaryantibody used was ALEXAFLUOR488™ (Molecular Probes, Invitrogen,Carlsbad, Calif.) conjugated. Images were obtained using a Zeissconfocal microscope with the laser at a maximum of 10% power. DHEfluorescence was exited at 543 nm and analyzed with a cutoff filterat >570 nm. Under these conditions, fluorescence analysis forparvalbumin and DHE could be used for quantification. #,*=statisticallysignificant compared to control with P<0.001 as analyzed by ANOVAfollowed by Tukey's test; n=6 animals/condition, or 5 cultures/conditionfor PFC and cultures, respectively.

The increase in ROS was widespread and not restricted to specificneuronal populations, as illustrated in FIG. 10, which illustratesconfocal images showing that ketamine induces ROS in vivo and in vitro.FIG. 10 illustrates representative confocal images from the experimentsdescribed in FIG. 9. The prefrontal cortex region analyzed correspondsto the prelimbic and infralimbic regions. Note that DHE fluorescence wasnot restricted to the PV-subpopulation of interneurons either in vivo orin the neuron culture system.

This increase in ROS production appears to be related to thedisinhibition of cortical circuitry as was described for rodents andnon-human primates upon exposure to the NMDA-receptor antagonists,phencyclidine and MK801. To analyze this possibility, primary neuroncultures were exposed to ketamine in the presence of the pan-GABA_((A))agonist muscimol (10 μM). Under these conditions, we observed a completeblock of ROS production and preservation of PV and GAD67 expression inPV-interneurons, these data graphically illustrated in FIG. 11. Theseresults confirm that the initial event upon ketamine exposure is adisinhibition of the system.

Co-exposure with muscimol prevents ketamine-mediated increase in ROS andloss of parvalbumin and GAD67 immunoreactivity; as shown by the datasummarized in FIG. 11. Primary cultures were exposed to ketamine for 24h as before in the absence or presence of the GABA_((A)) agonistmuscimol (10 μM). This concentration was chosen from previousobservations for its anti-apoptotic effects in this system. Sistercoverslips were treated similarly and DHE was added for the last hour oftreatment. Fixation and immunostaining was as described before. GAD67and parvalbumin immunoreactivity was quantified as described by Kinney(2006) J. Neurosci. 26:1604. *=Statistically significant with respect tocontrol at P<0.001 by ANOVA followed by Tukey's test. N=4 experimentsper condition.

Furthermore, these results also demonstrated that this disinhibition mayhave caused increased ROS. Of note, in primary cultures “disinhibition”can only mean increased glutamate, since by definition, these cultureslack neuronal inputs from outside the cortex.

It was next determined whether superoxide generation by ketamineexposure was due to activation of NADPH oxidase. The effects of a Noxinhibitor, apocynin, and a SOD-mimetic (C₃) were analyzed. Exposure ofprimary neuronal cultures to ketamine in the presence of C₃ preventedthe increase in ROS. More importantly, the Nox inhibitor apocynin alsoprevented the increase in free radical production, as shown by the datasummarized in FIG. 12, demonstrating that activation of Nox is involvedin the generation of superoxide upon administration of NMDA-receptorantagonists.

Data summarized in FIG. 12 demonstrates that a SOD mimetic (C₃), or theNox inhibitor apocynin (Apo) prevented ketamine-mediated superoxideand/or hydrogen peroxide production in cultures. Cultures were treatedwith ketamine as before in the absence or presence of 1 μM C3 or 500 μMapocynin for 24 h. DHE was added during the last hour of treatment.Cells were fixed as before and DHE fluorescence was analyzed by confocalmicroscopy. *=significantly different from control at P<0.001 by ANOVAfollowed by Tukey's test. N=4 experiments per condition.

We next analyzed the expression of Nox isoforms and subunits and foundthat cultures express mRNA for Nox2, Nox4, and p22^(phox), but not Nox1,Nox3 or Nox5 (data not shown). Expression of Nox2 and p22^(phox) proteinwas confirmed by Western blot and ICC, as illustrated in FIGS. 13A-13B,and was increased by ketamine. We did not see changes in Nox4 expressionafter ketamine treatment (not shown). Since only Nox2, but not Nox4 isinhibitable by apocynin at the concentrations used in our studies, thisdemonstrates that Nox2 is the isoform responsible for ketamine-mediatedROS, a result that can be confirmed in gp91^(phox)−/− mice.

Data illustrated in FIGS. 13A-13B shows Nox2 is expressed in cortex andketamine treatment increased its expression in vitro and in vivo.Ketamine treatment (0.5 μM, 24 h) increased the expression of Nox2 incultures FIG. 13A and also increased Nox2 and p22^(phox) in corticalparticulate fractions from ketamine treated animals FIG. 13B.

Use of Synaptosomal Preparations from Ketamine Treated Animals to Studythe Regulation of Nox Activity.

Synaptosomes, isolated nerve terminals whose axonal attachments havebeen severed by shear stress during homogenization, were used becausethey are a simple mammalian neuronal model in which functional cell-likeenvironments are physiologically maintained. In a synaptosome,mitochondria are present and supplied with substrates by themetabolic-machinery such that they produce ATP. Synaptosomal membranesretain ion pumps and channels as well as the components necessary forsynaptic vesicle exocytosis and recovery, and changes occurring in vivoare maintained in the isolated synaptosomal fractions.

We employed synaptosomal membrane preparations to study neuronal ROSproduction and to identify contributions by mitochondria and Noxisoforms. We also employed polarographic electrochemical determinationof Nox activity through NADPH-dependent oxygen consumption bysynaptosomes, and followed the parallel production of ROS bysynaptosomes using EPR spin-trapping spectroscopy. We assayed NADPHoxidase activity by following NADPH-dependent O₂ consumption in thepresence of Nox inhibitors.

FIG. 14A illustrates the experimental scheme where synaptosomes areisolated from mice brain cortex and split into two portions that aretransferred to the oxygraph chamber, where oxygen consumption ismonitored at different conditions; or to the EPR spectrometer tospin-trap and evaluate.

Consumption of oxygen by NOX and inhibition by apocynin is illustratedin the data summarized in FIG. 14B, which summarizes data showingdose-dependent inhibition of NADPH-stimulated O₂ consumption byapocynin; 200 μM apocynin was sufficient to block >90% of NADPH-inducedOz consumption (compare blue and black traces, and see inset). Comparingthe three oxygraph traces in FIG. 14B indicates that inclusion ofapocynin did not affect mitochondrial respiration or by inference, anyof the electron transport components. In FIG. 14B, approximately 5 mgsynaptosomal protein was incubated for 10 minutes at 37° C. with 200 μMdigitonin and different apocynin concentrations (0-200 μM) before theactivation of NOX was triggered by the addition of 5 mM NADPH. Viabilityof synaptosomal mitochondria in the presence of digitonin and apocyninwas assessed by respiration upon the addition of 10 mM malate and 10 mMpyruvate. F₀F₁-ATPase inhibitor oligomycin (4 μg/mL) halted oxygenconsumption and the maximal mitochondria uncoupling was established bythe addition of 0.5 μM CCCP as an indication of ‘healthy mitochondria”.

EPR Spectroscopy on Synaptosomal Preparations.

EPRS was used to measure ROS production by synaptosomes. For eachpreparation, 10 mg synaptosomal protein was mixed with 100 mM of theDIPPMPO spin-trap and the EPR spectra were recorded after 1 hr in theabsence or in the presence of Nox and/or mitochondrial substrates, c.f.all spectra in FIG. 15. Nitrone spin traps react with short-livedtransient radical species to form more stable nitroxide free radicaladducts that are easy to detect by EPRS. The signal can be interpretedthrough computer simulations to resolve contributions from differentradical species. The peak-heights or area-under-peak are parameters thatdepend on experimental conditions and the concentration of the freeradical species detected. By fixing various experimental and spectralconditions, we obtained EPR signals that correlate with free radicalconcentrations. Data demonstrated a 4-fold enhancement of the EPR signalin synaptosomes after addition of NADPH, demonstrating that

signal (marked by *) derives from NADPH oxidase. The source of

was further defined by showing that it was blocked by apocynin and didnot derive from mitochondria. Taken together, these results indicate thepresence of Nox-associated

production in synaptosomes.

The SOD mimetic and apocynin prevented ketamine effects onPV-interneurons in culture, these data are summarized in FIG. 15.Cultures were treated with ketamine for 24 h in the absence or presenceof C3 or apocynin. After fixation, quantitative parvalbumin and GAD67ICC was carried out. *=statistically significant at P<0.05 as comparedto control conditions by ANOVA followed by Tukey's test. N=4 experimentsper condition.

Synaptosomal Nox is an Active Source of Free Radicals.

EPR spectra recorded after 1 hr incubation of approximately (˜) 10 mgsynaptosomal protein isolated from mouse brain at 37° C. in the absenceof Nox or mitochondria substrates is shown in FIG. 16(i), in thepresence of 10 mM malate+10 mM pyruvate is shown in FIG. 16 (ii), or 200mM digitonin+5 mM NADPH is shown in FIG. 16(iii). The observed signalsare arising from DIPPMPO/superoxide adduct and matches that reported byChalier & Tordo (2002) J. Chem. Soc., Perkin Trans. 2, 2110-2117. Themixture was injected into the EPR cavity of Bruker e-scan benchtopspectrometer via a Teflon tube with inner diameter of ˜0.4 mm. The EPRsettings were, receiver gain 1×103, scan width 200 G centered at 3484.9G, modulation amplitude 4 G, time constant 5.16 ms, modulation frequency86 kHz, microwave power 5.04 mW, 5.24-s sweep time, and thespectrometer's operating frequency 9.784 GHz. Each spectrum was theaverage of 200-times accumulations.

Having characterized the activity of Nox in synaptosomes, we proceededto analyze the activity of the enzyme in synaptosomal fractions obtainedfrom ketamine-treated animals. Mice were treated with either saline orketamine (30 mg/kg) (4/group) on two consecutive days and sacrificed 18h after the last ketamine injection. Brains were immediately extractedand synaptosomes prepared as described above and analyzed for Noxactivity. Ketamine treatment in vivo induced a pronounced increase inNox activity in synaptosomes which was inhibited by apocynin. Mostimportantly, increased Nox activity did not affect mitochondrialfunction in synaptosomes, at least during the period of the experiment.

Involvement of Nox Activation in the Loss of Phenotype ofPV-Interneurons.

To further analyze the mechanism by which Nox is activated upon ketaminetreatment, and the possibility that superoxide derived from activationof Nox is involved in the loss of GABAergic phenotype ofPV-interneurons, we analyzed the effects of the SOD-mimetic C3 and theNox inhibitor apocynin on the effects of ketamine in parvalbumin andGAD67 immunoreactivity in the primary culture system. Preventingsuperoxide generation with apocynin, or inducing its dismutation withthe SOD-mimetic attenuated the decrease in parvalbumin and GAD67expression in PV-interneurons in culture. These demonstrated involvementof Nox activation in ketamine effects on PV-interneurons.

To confirm these results in vivo, mice were treated with ketamine in theabsence or presence of either apocynin or the brain-permeable SODmimetic (C₃). Ketamine reduced parvalbumin and GAD67 expression in thePFC, see FIG. 17, left. Treatment with C₃ or apocynin prevented the lossof parvalbumin in PV-interneurons and reduced DHE oxidation, see FIG.17, right. Furthermore, treatment with C₃ actually increased theexpression of the calcium-binding protein above control levels, a resultwe had already observed in the culture system.

FIG. 17 (Left panel): Animals were treated with ketamine (30 mg/kg. ip)applied on two consecutive days and sacrificed 18 h after the lastketamine injection. Coronal sections comprising the PFC (Bregma 2.0-1.3)were analyzed for parvalbumin and GAD67 expression in PV-interneurons asdescribed in Methods. FIG. 17 (Right panel): Animals were treated withapocynin in the drinking water for 1 week before the treatment withketamine, or during one month with the SOD-mimetic C₃ delivered bymini-pumps. and DHE was injected 30 min after the last ketamineapplication. The animals were deeply anesthetized, and perfusion fixedas described in the methods section. Coronal sections encompassing theprefrontal region were processed for IHC and analyzed for parvalbuminexpression and DHE fluorescence. N=5-6 animals per condition. * and #indicates statistical significance with respect to control at theindicated P values as analyzed by ANOVA followed by Tukey's multiplecomparisons test. Enlarged images are provided in the appendix section.

Role of the Pro-Inflammatory Cytokine Interleukin-6 in theKetamine-Mediated Increase of Nox Expression, Superoxide and/or HydrogenPeroxide Production, and Loss of Phenotype of PV-Interneurons.

Treatment with NMDA receptor antagonists has been shown to increase IL-6in plasma, and plasma levels of IL-6, in turn, correlate with the degreeof psychosis in schizophrenia patients. Therapeutic use of IL-6 forsolid tumors also induces psychosis, and interestingly, mice thatoverexpress IL-6 under the GFAP promoter demonstrate a loss ofPV-interneurons. Since pro-inflammatory cytokines, such as TNFα, IL-1β,and IL-6 are known to activate NFκB, and NFκB can regulate expression ofboth Nox2 and p22^(phox), we tested the possibility that IL-6 couldinduce Nox in vitro and in vivo, and further asked whether IL-6 might bea mediator in the ketamine-induced Nox expression and loss of phenotypeof the PV-immunoreactive GABAergic interneurons.

IL-6 increases superoxide in a Nox dependent manner, and reduces GAD67and parvalbumin in primary cultures, as summarized by the datagraphically presented in FIG. 18. Cultures were treated with IL-6 (10ng/ml) for 24 h in the absence or presence of apocynin (500 mM). Asbefore, dihydroethidium (DHE) was applied for the last hour (DHE detectssuperoxide and hydrogen peroxide). After fixation, ICC for PV and GAD67was carried out as described in methods and quantitative confocalmicroscopy was utilized for fluorescence analysis.

We treated primary neuronal cultures with IL-6 (10 ng/ml), aconcentration shown to modulate the activity of cortical neuronalcultures, and analyzed superoxide and/or hydrogen peroxide production aswell as immunoreactivity for parvalbumin and GAD67 in PV-interneurons.IL-6, when applied for 24 h, increased the levels of DHE oxidation anddecreased the immunoreactivity of GAD67 and parvalbumin, and theseeffects were prevented by the Nox inhibitor apocynin, as summarized inFIG. 18 (left).

To confirm that IL-6 mediates the increase in Nox2, we analyzed Nox2expression by immunocytochemistry and its activity by determination ofoxidized dihydroethidium (oxDHE). Primary cortical neurons exposed toIL-6 for 24 hours showed a pronounced increase in the expression ofNox2, as well as an increase in superoxide production (FIG. 18, left).The superoxide production was eliminated when apocynin was added alongwith IL-6, whereas Nox2 induction by IL-6 was not affected by theoxidase inhibitor. These results demonstrate that IL-6 is the downstreammediator of ketamine in the induction of Nox2.

The data of FIG. 18, left, demonstrates that IL-6 increases superoxideproduction and Nox2 expression in neurons. Neuronal cultures weretreated with IL-6 (10 ng/ml) in the absence (control) or presence of theNox2 inhibitor apocynin (0.5 mM) for 24 h. DHE (1 μg/ml) was addedduring the last hour of treatment. Images show the increase in Nox2immunoreactivity and oxidized DHE upon treatment with IL-6. Bar-graphsshow the results of quantification of oxidized DHE and Nox2 fluorescenceexpressed as % of control. (* P<0.001 by Tukey's test. ANOVA (oxDHE):P<0.001, F_(stat):40.712 _((oxDHE)); ANOVA_((Nox2)): P<0.001, F: 47.570,n=5 experiments). Data are means±SEM. Baseline intensities: DHE=21±4.7;Nox2=18.7±2.6.

When the cultures were treated with IL-6, but in the presence ofsub-threshold concentrations of ketamine, we found that IL-6 increasedthe effects of ketamine on parvalbumin and GAD67 immunoreactivity, assummarized in FIG. 18 (right). When primary neuronal cultures wereexposed to IL-6 (10 ng/ml for 24 h) we observed a decrease inparvalbumin and GAD67 in PV-interneurons (FIG. 18, right), demonstratingthat IL-6 is able to fully reproduce the ketamine effects we previouslyshowed in cultured neurons. IL-6 effects on PV-interneurons wereprevented by co-exposure to the NADPH oxidase inhibitor apocynin(4-hydroxy-3-methoxyacetophenone), indicating that, similar to ketamine,the interleukin effects were mediated by activation of Nox2-dependentNADPH oxidase superoxide production, as illustrated by the data of FIG.18.

The data of FIG. 18 (right) demonstrates IL-6 exposure leads to the lossof phenotype of PV-interneurons in primary neuronal cultures. Neuronalcultures were treated with IL-6 (10 ng/ml) in the absence (control) orpresence of the Nox2 inhibitor apocynin (0.5 mM) for 24 h. Fluorescenceconfocal images of representative fields depicting the expression ofparvalbumin (PV) and GAD67 in PV-interneurons. Bar-graph represents thequantification of fluorescence expressed as % of control. (* P=0.002, #P<0.001 by Tukey's test. ANOVA_((PV)): P<0.001, F: 11.860.ANOVA_((GAD67)): P<0.001, F: 24.912. n=4 experiments). Data aremeans±SEM. Baseline intensities: PV=135±32; GAD67=114±26.

It is possible that IL-6 causes the activation of Nox and increase insuperoxide in a fashion similar to the effects we observed for ketamine.So, in cultures we compared the effects of IL-6 and ketamine on Nox2expression in the presence and absence of the GABA_((A)) agonistmuscimol. Both ketamine and IL-6 induced expression of Nox2, butmuscimol only prevented the induction of the enzyme by ketamine, asillustrated in FIG. 20, this data indicating that IL-6 is downstream ofthe initial disinhibition caused by ketamine treatment

IL-6 Potentiates Ketamine Effects on PV-Interneurons.

Cultured neurons were treated with a subthreshold concentration ofketamine (0.05 mM) in the absence or presence of IL-6 (10 ng/ml) for 24h; this data is schematically summarized in FIG. 19. After fixation, ICCfor GAD67 and parvalbumin was carried out as described by Kinney (2006)J. Neurosci. 26:1604. * indicates statistically significant with respectto control at P<0.05, and # indicates significantly different withrespect to ketamine or IL-6 alone at P<0.05 by ANOVA followed by Tukey'stest. N=3 cultures per condition.

Muscimol prevents only ketamine-mediated induction of Nox2 in primarycultures; this data is schematically summarized in FIG. 20. Cultureswere treated with ketamine (0.5 μM) or IL-6 (10 ng/ml) in the absence orpresence of muscimol (10 mM) for 24 h. Nox immunoreactivity was analyzedby ICC using anti-Nox and anti-MAP2 double immunofluorescence. Valuesare expressed as percent of control conditions (no treatment). *indicates statistical significance with respect to control conditions atp<0.05 by ANOVA followed by Tukey's test. N=200 cells across threeexperiments.

We therefore tested the possibility that upon treatment with ketaminethere is an induction of IL-6 expression, which would in turn beresponsible for Nox induction. RT-PCR performed on RNA obtained fromketamine-treated cultures showed increased IL-6 RNA. As illustrated inFIG. 21, ketamine induced IL-6 mRNA expression in cultures. Cultureswere treated with ketamine (0.5 μM) for varying periods of time and mRNAwas extracted using TRIZOL™ (Invitrogen, Carlsbad, Calif.), see Methods,below. RT-PCR was performed using primers specific for murine IL-6 andfor GAPDH as internal control. IL-6 mRNA was significantly increasedalready at 4 h of ketamine exposure. The mRNA levels decreased after 6 hto levels that were above control after 24 h.

These results led us to believe that NMDA-receptor antagonists, throughinhibition of PV-interneuron function, trigger a mild inflammatoryreaction in brain which increases brain IL-6 levels and thereforeneuronal expression of Nox. Conversely, since increased IL-6 plasmalevels are a consistent finding in schizophrenic patients, andNMDA-receptor antagonists increase plasma levels of IL-6 applied i.c.v.in rodents, we wanted to determine whether IL-6 administeredperipherally intraperitoneally (i.p) would have any effect on neuronalNox expression and/or loss of PV-interneuron phenotype. Animals weretreated on two consecutive days with 5 μg/kg IL-6 or saline, andsynaptosomal preparations were prepared and analyzed. We observed asignificant induction of Nox activity, as illustrated in FIG. 22,demonstrating that plasma IL-6 can have CNS effects.

To generate the data illustrated in FIG. 22, mice (4 animals percondition) were treated with IL-6 (5 ug/kg) on two consecutive days atthe same time of the day. Synaptosomes were prepared after 22 hours (h)of the last injection, NADPH-dependent oxygen consumption was analyzedin the absence or presence of apocynin, 150 uM. The apocynin effectclearly shows that as occurred with ketamine treatment, where IL-6induces preferentially Nox2 in the brain.

We also observed increased apocynin-inhibitable DHE oxidation, andincreased Nox2 expression by IHC (data not shown). The question ofwhether IL-6 crosses the BBB directly, or triggers secondary eventswhich lead to Nox induction is not addressed by this experiment,although the latter is more likely based on previous studies showingthat LPS administered to IL-6−/− mice fails to disrupt learning andmemory, although plasma cytokine levels are high. However, thesefindings do demonstrate that elevated plasma IL-6 can result in theinduction of brain Nox, superoxide and/or hydrogen peroxide productionand loss of the GABAergic phenotype of PV-interneurons.

Behrens (2007) Science 318:1645-1647; showed that exposure tosub-anesthetic levels of ketamine on two consecutive days induces apronounced increase in brain superoxide through activation ofNADPH-oxidase, and that this leads to the loss of phenotype ofPV-interneurons in prefrontal cortex.

In this study, the effects of ketamine on PV-interneurons in theprefrontal region were observed only after exposure on two consecutivedays, and not present 24 h following a single exposure, as illustratedin FIG. 23, as previously reported for rat by Cochran (2002) Synapse46:206-214. FIG. 23 graphically illustrates data showing the slowreversal of ketamine effects on PV-interneurons in vivo. C57BL/6 mice (3month-old males) were treated with ketamine (30 mg/kg i.p.) on one ortwo consecutive days as described by Behrens et al., 2007, supra.Animals were sacrificed either 24 hr after a single injection or 1, 3,or 10 days after the second ketamine injection. Coronal brain sectionscomprising the prelimbic region were analyzed by fluorescenceimmunohistochemistry for parvalbumin (PV) and GAD67, and expressed aspercent of saline treated controls. A slow increase in fluorescenceintensity for both proteins is observed starting at 3 days after thesecond ketamine injection (* statistically significant with respect tosaline at P<0.05; # statistically significant with respect to 2 days ofketamine at P<0.05. As determined by one way ANOVA followed by Tukey'stest. ANOVA_((PV)): P<0.001, F: 16.344; ANOVA_((GAD67)): P<0.001, F:20.926. n=15 animals for saline and 5 animals per time point. Each timepoint consisted of 5 saline and 5 ketamine treated animals. Since nodifferences were observed for the saline treated mice, these values werecombined. Data are means±SD. Mean fluorescence intensity for saline:PV=160.6+/−13.3; GAD67=110.4+/−8.6.

Furthermore, as previously shown in microdialysis studies of rats 24 hafter exposure to a single injection of ketamine by Zuo (2007) PharmacolBiochem Behav. 86:1-7, we did not observe increase in DHE oxidation inthe prelimbic region of mice 24 h after a single injection of ketamine(not shown).

These results support the conclusion that repeated exposure to NMDA-Rantagonists is required to produce persistent changes in PV-interneuronphenotype and function; see e.g., Cochran (2003) Neuropsychopharmacology28:265-275; Keilhoff (2004) Neuroscience 126:591-598; Rujescu (2006)Biol Psychiatry 59:721-729.

To test for the enduring effects of the two-day ketamine treatment onthe loss of phenotype of PV-interneurons, adult male C57BL/6 mice weretreated with ketamine (30 mg/kg) on two consecutive days and thePV-interneuronal population in the prelimbic region was analyzed on days1, 3, and 10 after the last ketamine injection. As previously described(e.g., by Behrens et al., 2007, supra), a pronounced decrease in theexpression of PV and GAD67 in PV-interneurons was observed one day afterwithdrawal; see FIG. 23. A slow reversal of this process was observed,although it still remained significant with respect to saline treatedanimals 10 days after withdrawal. The decrease was specific for thePV-interneuronal population, as demonstrated by the lack of effects ofthe 2-day ketamine treatment on the levels of calbindin (Meanintensity±SD: Saline=215.6±32.1; GAD67: 30.1±14.3, Ketamine=231.6±25.6,GAD67=44.2±14.5. ANOVA_((CB)): P=0.477, F=0.558. ANOVA_((GAD67)):P=0.588, F=0.319. n=6 animals per condition) and calretinin (Meanintensity±SD: Saline=133.6±35.6; GAD67: 30.1±14.3, Ketamine=139.4±28.7,GAD67=44.2±14.5. ANOVA_((CR)): P=0.786, F=0.079. ANOVA_((GAD67)):P=0.922, F=0.01. n=6 animals per condition).

To confirm the role of Nox2-dependent NADPH oxidase (Nox2) in thesuperoxide mediated loss of phenotype of PV-interneurons we exposedadult Nox2-deficient (gp91^(phox)−/−) male mice to ketamine (30 mg/kg)on two consecutive days, and injected dihydroethidium (DHE) 30 min afterthe last ketamine treatment to measure superoxide production asdescribed by Behrens et al., 2007, supra.

Analysis of the prelimbic region showed that deletion of Nox2 preventedthe increase in superoxide induced by ketamine, as shown by the datagraphically illustrated in FIG. 24A, and protected the phenotype ofPV-interneurons, as shown by the data graphically illustrated in FIG.24B. The data of FIG. 24 demonstrates the absence of ketamine effects inthe PFC of Nox2 knockout mice. Three month old gp91^(phox)−/− weretreated with ketamine (30 mg/kg i.p. on two consecutive days) followedby DHE injections. Coronal sections comprising the prelimbic andinfralimbic regions were analyzed for (FIG. 24A) oxidized DHE, and (FIG.24B) parvalbumin (PV) immunofluorescence. Fluorescence intensity isexpressed as percent of saline treated C57BL/6 animals. (A: ox-DHE: *, #significant with respect to saline C57BL/6. * P<0.001, #P=0.026 byTukey's test. ANOVA: P<0.001, F: 26.782; B: PV: * P<0.001 with respectto saline C57BL/6. ANOVA: P<0.001, F: 11.555). Data are means±SD. Meanfluorescence intensity for saline C57BL/6 control: ox-DHE=9.8+/−1.5;PV=147.2+/−23.3.

FIG. 25 graphically illustrates data from neuronal cultures exposed toketamine and IL-6, which shows that blocking activity of thetranscription factor NFκB using SN50 blocks induction and activation ofNox2, as assessed by DHE oxidation. The NFkB inhibitor SN50 blocks IL-6induced superoxide production in neuronal cultures. Cultures wereexposed to IL-6 with or without SN50 and superoxide production (DHEoxidation) was assessed 4 hours later. Inhibition of NFkB blockedinduction and activation of Nox2 in neurons by IL-6.

These results confirm the specific role of Nox2-dependent superoxideproduction in the loss of phenotype of PV-interneurons caused byketamine exposure. Increased basal level of superoxide production ingp91phox−/− animals were previously observed, and attributed todevelopmental compensatory mechanisms that lead to increased expressionof other Nox subunits; see e.g., Byrne (2003) Circ Res 93:802-805; Liu(2007) Can J Neurol Sci 34:356-361.

We also observed an increased basal level of DHE oxidation in brains ofNox2-deficient animals, as illustrated in the data summarized in FIG.24A. However, this level of superoxide production was not sufficient toaffect PV-interneurons, as illustrated in the data summarized in FIG.24A. These results give strong support to a specific role ofNox2-dependent activation in the effects of NMDA-R antagonists onPV-interneurons.

Ketamine Exposure Induces IL-6 Expression.

To directly examine whether ketamine exposure induced the expression ofthe cytokine in neurons, we exposed primary cortical cultures toketamine and analyzed IL-6, IL-1 and TNF mRNA at different time pointsduring the 24 hours exposure. PCR amplification of reverse transcribedmRNA showed that ketamine exposure induced a sustained increase only inIL-6 transcript; as graphically illustrated by the data in FIG. 21,without affecting the levels of other pro-inflammatory cytokines. Thelevel of IL-6 mRNA remained significantly elevated with respect tocontrol conditions 24 hours after ketamine (180±18.1%, P=0.01). In FIG.21 primary neuronal cultures were exposed to ketamine (0.5 μM) for thetimes indicated and the abundance of IL-6 mRNA was determined by PCRusing specific primers after reverse-transcription of mRNA obtained fromthe cultures. Values for IL-6 mRNA abundance were obtained afternormalization by the expression of GAPDH mRNA in the samples. (*indicates significance with respect to control conditions(P_((3 h))=0.009, P_((6 h))=0.001) by Tukey's test. ANOVA: P=0.001, F:46.950. n=3 experiments per time-point).

Primary neuronal cultures also were exposed to ketamine (0.5 μM) for thetimes indicated and the abundance of IL-6, IL-1, and TNF mRNA weredetermined by PCR using specific primers after reverse-transcription ofmRNA obtained from the cultures. Values for mRNA abundance were obtainedafter normalization by the expression of GAPDH mRNA in the samples;significance with respect to control conditions at P<0.001 determined byTukey's multiple comparisons test. ANOVA: P=0.001. F: 46.950.

To test if glial cells were responsible for the increase in IL-6 uponketamine exposure, we applied the NMDA-R antagonist to neurons in theabsence of the astrocytic layer, and analyzed the PV-interneuronalpopulation 24 h later. Ketamine produced a similar increase in DHEoxidation and loss of phenotype of PV-interneurons in the presence orabsence of the astrocytic layer, as graphically illustrated by the dataof FIG. 26, demonstrating that if IL-6 mediates these effects, it mustbe of neuronal origin.

To generate the data of FIG. 26, primary neuronal cultures were grown onglass coverslips with “feet” as described by Kinney (2006) J. Neurosci.26:1604. After 21 days of development in vitro, the cultures weretreated with ketamine (0.5 mM for 24 h) in the presence or absence ofthe astrocytic layer. For this, the coverslips containing neurons wereseparated from the astrocytic layer by transfer of the coversliptogether with its media into an empty well. DHE was added for the lasthour of treatment as described by Behrens et al., 2007, supra. Aftertreatment, neurons were fixed and processed for immunofluorescence fordetection of either PV or GAD67 or for oxidized DHE. *, # indicatesstatistical significance with respect to control conditions at P<0.001by ANOVA followed by Tukey's test. ANOVA(PV): P=0.003. F: 7.569;ANOVA(GAD67): P<0.001, F: 10.103; ANOVA(oxDHE): P<0.001, F: 94.583.n=3-5 experiments per condition. Data are means±SEM. Baselineintensities: PV=210±32; GAD67=195±26.

To confirm this hypothesis, we applied IL-6 blocking antibodies, asdescribed e.g., by Smith (2007) J. Neurosci. 27:10695-10702, during the24 hours exposure of primary neurons to ketamine in the absence of theastrocytic layer. Blocking IL-6 with two different antibodies completelyprevented ketamine effects on PV-interneurons, as shown by the datagraphically illustrated in FIG. 27A; and also the increase insuperoxide, as shown by the data graphically illustrated in FIG. 27B,indicating that IL-6 is the downstream mediator of ketamine effects onNox2 induction and activation.

For FIGS. 27A-27B: primary neuronal cultures were exposed to ketamine inthe absence of the astrocytic monolayer and in the presence of ananti-mouse IL-6 blocking antibody produced in goat (anti-mIL-6). FIG.27A: Increasing concentrations of anti-mIL-6 prevented the decrease inparvalbumin (PV) and GAD67 after 24 h of ketamine exposure. Bar graphshow results for fluorescence quantification of both antigens inPV-interneurons expressed as % of control. *,** P=0.006 and 0.028respectively; ^(#,##) P<0.001 by Tukey's test. ANOVA_((PV)): P=0.002, F:4.564, ANOVA_((GAD67)): P<0.001, F: 27.512; n=4 experiments percondition. Baseline intensities: PV=165±30; GAD67=127±28. FIG. 27B:Neuronal cultures were treated as in A, and DHE was added for the lasthour of treatment. After fixation, the coverslips were processed forimmunocytochemistry for parvalbumin (PV, green). Bar graph show resultsfor oxidized DHE fluorescence (red) intensity analysis in all neuronsincluding PV-interneurons. (* P<0.001 with respect to control and **P<0.001 with respect to ketamine by one way ANOVA followed by Tukey'stest. ANOVA: P<0.001, F_(stat): 46.415. n=3 experiments per condition).Baseline intensities: DHE=25.4±5.4.

For these experiments, two different blocking antibodies, produced indifferent species, were used. The blocking capacity of these twoantibodies differ by a factor often (as described by manufacturer), anda similar difference was observed when blocking ketamine effects, asgraphically illustrated by the data shown in FIGS. 27 and 28.

For FIG. 28: primary neuronal cultures were exposed to ketamine in theabsence of the astrocytic monolayer and in the presence of an anti-mouseIL-6 blocking antibody produced in rat (anti-mIL-6). Increasingconcentrations of anti-mIL-6 prevented the decrease in parvalbumin (PV)and GAD67 after 24 h of ketamine exposure. Bar graph show results forfluorescence quantification of both antigens in PV-interneuronsexpressed as % of control. *,# P<0.001 with respect to control byTukey's multiple comparisons test. ANOVA_((PV)): P<0.001, F: 28.727;ANOVA(GAD67): P<0.001, F: 39.684. n=3 experiments per condition.

The data graphically illustrated in FIGS. 29A-29B show that ketaminedoes not lead to loss of GABAergic phenotype of PV-interneurons inIL-6−/− mice. To assess whether IL-6 and other inflammatory cytokineswere induced in brain after ketamine exposure we analyzed the levels ofmRNA for IL-6, IL-1β and TNFα, as previously shown for cultured neurons.Exposure to ketamine on two consecutive days only increased the levelsof IL-6 mRNA, as illustrated in FIG. 29A, without affecting mRNA levelsof IL-1 or TNF.

To further assess the role of IL-6 in ketamine effects in vivo, weexposed IL-6-deficient mice to ketamine on two consecutive days, andanalyzed the PV-interneuronal population in the prefrontal region, aswell as the activity of Nox2-dependent superoxide production by DHEoxidation. Lack of in vivo production of IL-6 prevented ketamineactivation of NADPH oxidase, as determined by the diminished DHEoxidation in the IL-6-deficient mice (FIG. 29A). Moreover, the phenotypeof PV-interneurons in the prefrontal region was preserved in theIL-6-deficient animals (FIG. 29B). These results demonstrate that CNSproduction of IL-6 is necessary and sufficient for the increase inNox2-dependent NADPH oxidase activity that leads to the loss ofphenotype of PV-interneurons observed after ketamine exposure.

In FIGS. 29A-29B, illustrating data showing CNS production of IL-6mediating ketamine effects on Nox and PV-interneurons in vivo: FIG. 29A:animals were treated with saline or ketamine (30 mg/kg) ontwo-consecutive days and the brains extracted for mRNA preparation 24 hafter the last ketamine injection. The abundance of IL-6, IL-1β, andTNFα mRNA was determined by PCR using specific primers afterreverse-transcription of mRNA obtained from forebrains. Values for mRNAabundance were obtained after normalization by the expression of GAPDHmRNA in the samples. (* indicates significance with respect to controlconditions at P=0.012 by ANOVA followed by Tukey's test. F: 12.775, n=4animals per condition). In FIG. 29B, three month old C57BL/6 (wt) orIL-6-deficient (IL-6(−/−)) male mice were treated with ketamine (30mg/kg) on two consecutive days, followed by DHE, as described by Behrenset al., 2007, supra. Coronal sections comprising the prelimbic andinfralimbic regions were analyzed by immunohistochemistry forparvalbumin (PV) and oxidized DHE fluorescence. Ketamine produced asubstantial increase in oxidized DHE in wild type mice but not inIL-6(−/−) animals. The loss of parvalbumin expression induced byketamine was prevented in the IL-6(−/−) animals. (*=oxDHE wt-saline vswt-ketamine P<0.001; **=oxDHE wt-ketamine vs IL-6(−/−) P=0.001 byTukey's test. ANOVA_((oxDHE)): P<0.001, F: 18.577. #=PV wt-sal vswt-ketamine at P<0.001; ##=PV wt-ketamine vs IL-6(−/−) P<0.001 byTukey's test. ANOVA_((PV)): P<0.001, F: 30.184. n=4 animals percondition). Data are means±SD. Mean fluorescence intensity for saline:wild type, PV=111.6+/−9.3; ox-DHE=10.1+/−2.5; IL-6(−/−),PV=101.7+/−10.2; ox-DHE=11.7+/−3.2.

The data graphically illustrated in FIGS. 30A-30B shows that IL-6directly activates NADPH oxidase. Superoxide production by live neurons,as analyzed by electron paramagnetic resonance (EPR), increased rapidlyafter ketamine exposure (FIG. 30). To confirm that this effect ofketamine was mediated by IL-6, a blocking antibody against IL-6 wasapplied during the exposure to ketamine and the activity of Nox wasanalyzed by EPR in live cultures as before. Blocking IL-6 action withthe antibody prevented the activation of Nox by ketamine (FIG. 30A).Moreover, to further test whether IL-6 triggers the signaling cascadesthat activate the oxidase, synaptosomal preparations were exposed toIL-6 (100 ng/ml) and superoxide production was assayed by EPR. IL-6produced a small but significant increase in superoxide that wascompletely blocked by co-exposure to apocynin, demonstrating that it wasproduced by Nox2-dependent NADPH oxidase (FIG. 30B).

In FIGS. 30A-30B, illustrating data showing that ketamine-induced IL-6release directly activates Nox. A: EPR assessment of superoxideproduction in live cultures upon treatment with ketamine (0.5 μM).Primary cultures were exposed to ketamine for the times indicated in theabsence or presence of an anti-mouse IL-6 blocking antibody produced inrat (anti-IL-6, 0.1 μg/ml). At the indicated times, the coverslips weretransferred to a quartz chamber and superoxide production was followedby EPR spectroscopy using the spin-trap DIPPMPO. Ketamine induced arapid increase in superoxide signals that were significantly reduced bythe blocking antibody (*=significant with respect control, P=0.03 and0.0002 for 1 h and 3 h, respectively. #=significant with respect toketamine, P<0.05 by Tukey's test of multiple comparisons. 2-way ANOVA:P=0.002, F=7.786. n=3-6 experiments per condition). FIG. 30B: IL-6 (100ng/ml) increased basal NADPH oxidase activity in forebrain synaptosomesisolated from 3 month-old C57BL/6 male forebrains. IL-6 waspre-incubated with synaptosomal preparations for 5 minutes beforetriggering oxidase activity by addition of substrate, NADPH. Apocynin(0.4 mM) was applied 5 min before IL-6. Accumulation of superoxideduring the first 6 min was analyzed using the spin trap DEPMPO. Data aremeans±SEM. *P<0.001 control vs. IL-6 and ^(#)P<0.001 apocynin treatedvs. no apocynin by ANOVA and Tukey's post-hoc test, F: 55.8, n=5-7experiments per condition.

General Methods Example 2

Maintenance of Mice, and In Vivo Administration of Ketamine, NoxInhibitors, SOD Mimetic Inhibitors, IL-6.

All mice for these studies are housed in the barrier facility at UCSD.Pathogen-free C57BL6 mice will be obtained from Jackson Labs, and the PImaintains breeding colonies of gp91phox−/− and IL-6−/− mice. gp91phox−/−mice are maintained on autoclaved water and are handled with steriletechnique during weaning. All animal studies have been approved by theAnimal Care Program at the University of California, San Diego, and arein accordance the PHS Guide for the Care and Use of Laboratory Animals,USDA Regulations, and the AVMA Panel on Euthanasia. The Nox inhibitor,apocynin (5 mg/kg/day) and the SOD mimetic, C₃ (a SOD inhibitor), 1mg/kg/day, will be given in the drinking water for 7 or more days, withan assumed intake of 13 ml H₂O/mouse/day.

In our preliminary studies C₃ was given through mini-pumps. Since wehave shown that it can be given in the drinking water also (Quick etal., 2006), we prefer this way of delivery in the future to avoid animalsurgery. Intracerebral (i.c.v.) injection of IL-6 or ketamine will beperformed only if needed using a mouse stereotax and establishedcoordinates. Intraperitoneal IL-6 (5 μg/kg), a dose established from ourprevious dose-response studies.

Analysis of Superoxide and/or Hydrogen Peroxide Production by ConfocalImaging of In Vivo Dihydroethidium (DHE) Oxidation.

Mice will be injected intraperitoneally (i.p.) with dihydroethidium.Briefly, two serial i.p. injections of freshly prepared dihydroethidium(27 mg/kg) are given at 30 minute intervals. Eighteen hours later, miceare anesthetized with inhaled halothane, and are perfused intracardiallywith cold saline followed by 4% paraformaldehyde in PBS. Brains areremoved and post-fixed in 2% paraformaldehyde for >24 h. Followingfixation, brains are cut into 50 μm coronal sections and co-labeled withthe appropriate primary and secondary antibodies for fluorescencevisualization. Slices are mounted and evaluated for fluorescence fromthe DHE oxidation product using Ex λ 568 nm, Em λ>590 nm on a LSM510META™ multiphoton laser confocal microscope (Karl Zeiss, Inc.). First,an image is taken in the fluorescence channel of the ICC-fluorophor andthen the channel is switched to image fluorescence from DHE oxidation.Autofluorescence is determined in animals which did not receive DHEinjections, but which are processed similarly to injected animals. Usingthe image pairs for each field and MetaMorph software, an analyst blindto the group circles the outline of each ICC-labeled cell and thenswitches to the DHE oxidation image. The average fluorescence intensityfor each cell of interest is logged, and values averaged to determinethe mean fluorescence/cell.

Immunohistochemistry (IHC) and Immunocytochemistry (ICC).

Mice are anesthetized and perfused as above. Coronal sections, at 50 μmthickness are treated with 1% sodium borohydride for antigen retrieval,washed, and blocked in 10% normal serum overnight. Slices are incubatedwith primary antibodies (Abs) in 2% normal serum at 4° C. overnight,washed in PBS, and incubated in secondary Abs conjugated to ALEXAFLUOR™dyes (488 and 568) for 1 hour at room temperature. When analyzing DHEoxidation and a specific Ab staining, secondary Abs are alwaysALEXAFLUOR488™ dyes conjugated. For ICC in cultures, coverslips arewashed by immersion in PBS, and fixed in ice-cold 4% paraformaldehydefor 30 min, and then incubated for 10 min at room temperature in PBScontaining 0.25% Triton X-100. Non-specific sites are blocked byincubation in PBS containing 10% serum (goat or horse). For doubleimmunostaining, the coverslips are incubated in 2% normal goat serumcontaining mAb against GAD67 (1:1000, Chemicon), Nox2 (1:200) or p47phox(1:50), rabbit polyclonal Ab against Parvalbumin (1:3000, Swant,Bellinzona, Switzerland), MAP2 (1:1000, Chemicon), or p22^(phox) (1:300.Santa Cruz), and incubated for 1-2 h at 37° C. Specific binding isdetected by incubation for 45 min at room temperature with a 1:1000dilution of secondary Abs conjugated to ALEXAFLUOR™ dyes (568: red, 488:green. Molecular Probes).

Fluorescence Quantification of IHC and ICC.

The settings on the confocal microscope are maintained constant for eachseries of experiments to allow images to be analyzed and compared bydensitometry. For cell imaging, each slice is imaged across theprelimbic and infralimbic regions between Bregma 1.3 and 2.0. Six slicesare analyzed per animal by taking 3×8 image stacks (corresponding to 1.4μm) of the region with a 10× APOFLUOR™ objective encompassing the wholePFC (18×8 images per animal). All PV-neurons in the images are analyzedfor their parvalbumin and GAD67 content. For analysis of overallneuronal population in primary cultures, coverslips are scanned toobtain 200-400 neurons (approx. 26-30 images captured per coverslip percondition using a 40× water immersion objective). Each image analyzedconsists of a stack of 16 0.2 μm Z-stage images taken from the base ofthe neurons and across 3.2 μm depth. When analyzing PV-interneurons, thecoverslips are scanned to obtain images as before but for all thePV-interneurons in the coverslip.

Analysis of Nox Proteins by Western Blot.

Preparation of samples and subcellular fractionation is carried out asdescribed below. For Western blotting, samples are prepared for SDS-PAGEusing standard procedures. After SDS-PAGE on 10-12% acrylamide gels,proteins are transferred to a nitrocellulose membrane, blocked inTris-Buffered Saline TWEEN-20™ (TBST) with 5% milk, and incubated withAbs to Nox(s) or subunit proteins at 4° C. overnight. We haveestablished conditions for the following Abs: monoclonals 44.1(p22^(phox); 1:1000) and 54.1 (Nox2; 1:1000), Nox4 polyclonal (1:1500),and p47phox monoclonal (1:50, Santa Cruz). After incubation withhost-appropriate secondary Abs, the membranes are washed and developedwith ECL (Pierce, Inc.).

Isolation of Synaptosomes.

For each preparation, pooling of two forebrains was found to providesufficient protein (10-15 mg) to run one oxygraph measurement and oneEPR experiment. Mice are euthanized by exposure to a lethal dose ofinhaled halothane, followed by cervical dislocation and brains arerapidly removed and placed onto ice-cold isolation buffer (0.32 Msucrose, 1 mM EDTA, 10 mM Tris-HCl buffer, pH 7.4, 10 mM glucose). Usinga glass-dounce homogenizer, the cortex is minced and homogenized inisolation buffer. The homogenate is centrifuged at 3100 rpm for 3 min at4° C., and the supernatant is collected; the pellet is re-homogenized inhalf the volume of isolation buffer and centrifuged again. Thesupernatants are pooled, and mixed with PERCOLL™ to a finalconcentration of 15%. The sample is layered onto a gradient of 23% and40% PERCOLL™. The fractions are separated by centrifugation at 16.000rpm for 5 minutes. The uppermost band is extracted, rinsed in theisolation buffer, centrifuged and resuspended in synaptosome buffer (120mM NaCl, 4.7 mM KCl, 2.2 mM CaCl₂, 1.2 mM MgCl₂, 25 mM HEPES, 1.2 mMMgSO₄, 1.2 mM KH₂PO₄, 10 mM glucose).

Oxygen Consumption Studies (Oximetry).

O₂ consumption studies on synaptosomes are carried out. O₂ utilizationis measured using an oxygen Clark-type electrode, OXYGRAPH™ (Hansatech,UK) with OXYGRAPH™ software. Studies were carried out to optimize theconcentration of synaptosomal protein required, and to confirm stabilityand viability of synaptosomes and their mitochondria for up to 6 h.

Superoxide Detection by Electron Paramagnetic Resonance (EPR)Spectroscopy:

After incubation of the reaction mixture containing 5 mg synaptosomalprotein, 70 mM DIPPMPO(5-(diisopropoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide, AlexisBiochemicals, San Diego, Calif.), and appropriate combinations of thesubstrates/inhibitors for 1 hr at 37° C., the mixture was injected intothe EPR cavity of a Bruker ESCAN™ (eScan) Benchtop spectrometer (BrukerBioSpin, MA, USA) through a gas-permeable Teflon tube. The EPR settingswere: receiver gain, 1×10³, scan width, 200 G centered at 3484.9 G,modulation amplitude 4 G, time constant 5.16 ms, modulation frequency 86kHz, microwave power 5.04 mW, 5.24-s sweep time, and the spectrometeroperating frequency was 9.784 GHz.

Preparation of Cortical Cell Cultures.

One-step neuronal-glial cultures—(used for confocal imaging andbiochemistry). Cultures are prepared from fetal (E14-15) Swiss Webstermice as described (Kinney et al., 2006). Briefly, cortices are dissectedfrom the rest of the brain, placed in 5 ml of growth media, whichconsists of media stock (MS: Eagle's Minimal Essential Media minusglutamine) with the addition of 20 mM glucose, 26.2 mM NaHCO₃, 2 mMglutamine, 5% fetal calf serum, 5% horse serum. The tissue is thentriturated using a 5 ml pipette, and cell suspensions are diluted to0.15 cortices/ml and plated onto poly-lysine coated coverslips. Whenglia has reached confluency, proliferation is halted by addition of 10μM cytosine arabinoside (AraC) for 48 h. Cultures are fed biweekly withgrowth media (MS with 10% horse serum), and used for experiments atDIV21-28. Dissociated neuronal cultures—(used for confocal microscopy)Cortical neurons are cultured at low density from the same E14-15dissections described above, following a slightly modified procedureform that described for rat hippocampal cultures which we substitutedovoalbumin for 2% horse serum in the media, since we discovered thatmouse astrocytes do not survive in the presence of this protein.Briefly, neurons are seeded on glass coverslips to which paraffin feetwere added, after cell attachment the coverslips are flipped on top ofthe astrocytes grown in MS/N2.1 media (MS plus 1× N2.1 supplements(Gibco), 2% horse serum, 2 mM L-glutamine, 1 mM pyruvate, and 12 mMglucose) as described e.g. by Kinney, et al. (2006) J. Neurosci.26:1604-1615. Five (5) μM cytosine arabinoside is immediately added tohalt the grown of non-neuronal cells. These co-cultures are maintainedin MS/N2.1 media for 21 days. Most neurons develop the characteristicmorphology of pyramidal cells. Immunostaining with αCaMKII antibodiesand GAD67 antibodies confirmed that 80-90% of the population ispyramidal neurons and 10-20% is GABAergic neurons.

Quantitative PCR.

qPCR for cytokines including IL-6, IL-2, and TNFα will be performed atthe Gene-Array Core Facility at UCSD using published qPCR primer sets.The results are normalized by the expression levels of the referencegene, GAPDH, which is quantified simultaneously with the target.

Statistical Analysis.

All intensity values were normalized by the mean obtained for thecontrol (primary cultures) or saline (in vivo) conditions for eachexperiment, processed in parallel with the experimental group, andexpressed as a percent of this mean. To obtain the meanfluorescence/cell/animal, % values were averaged across the six slicesof the same animal (or experiment in the case of primary cultures incoverslips), and the mean fluorescence intensity/cell/animal (or perexperiment in the case of primary cultures) was used to calculate themean and standard deviation per group. These were then used forstatistical analysis using SigmaStat software. Values obtained perexperiment were analyzed by one-way ANOVA followed by Tukey's post-hoctest for multiple comparisons. ANOVA results were considered significantwhen P<0.05.

Process for the Removal of Contaminants from Preparations of MalonicAcid Derivatives of Fullerene C60

In one embodiment, C60 fullerene derivatives (e.g., C₃, or tris malonicacid C60) or other malonic acid derivatives are used to practice thisinvention, e.g., as therapeutic agents for the clinical applicationsdescribed herein. While the invention is not limited by any particularmechanism of action, in one embodiment, the C60 fullerenes (e.g., C3, ortris malonic acid C60) or other malonic acid derivatives act assuperoxide dismutase mimetics, thereby augmenting the action ofendogenous SOD to decrease the amount of superoxide, thereby having acytoprotective effect. In one embodiment, the class of compoundscomprising malonic acid derivatives, including C₃ (tris malonic acidC60), are used to practice this invention; these compounds arecytoprotective in cell culture and animal models of disease, and are inpreclinical testing.

Any method known in the art can be used to purify and/or prepare C60fullerene derivatives (e.g., C₃, or tris malonic acid C60) or malonicacid derivatives to practice this invention, including for example thepurification and scale-up synthesis protocols for C₃ as described bye.g., U.S. Pat. No. 6,538,153, Hirsch, et al., describing methodscomprising steps of forming macrocyclic malonate compounds, includingthe tris malonic acid C60; or as described in U.S. Pat. No. 7,070,810,Hirsch, et al., describing amphiphilic substituted fullerenes andfullerenes comprising a fullerene core and a functional moiety, andmethods for making them; or as described by C. Bingel (1993) Chem. Ber.126:1957. The Bingel reaction is a popular method in fullerene chemistrywhere the malonate is functionalized with a halide atom in a mixture ofbase and tetrachloromethane or iodine; the reaction can take place withester groups replaced by alkyne groups in dialkynylmethanofullerenes.

C60 fullerene derivatives (e.g., C₃, or tris malonic acid C60) or othermalonic acid derivatives to practice this invention also can be preparedand/or purified as described herein, where this invention provides a newmethod for purifying C60 fullerene derivatives (e.g., C3, or trismalonic acid C60) or other malonic acid derivatives. In one embodimentof this method, preparations of C₃ are prepared in water as a pH-neutralsalt. Sodium hydroxide was used, but other salt preparations can be usedin alternative embodiments. After incubation for a period of time, thetoxic, waxy contaminant begins to precipitate, and can then be removedby high-speed centrifugation, filtering, appropriate columnchromatography, or other techniques. Any residual volatile contaminantcan be removed by vacuum distillation to produce a dried powder whichcan be re-dissolved in water to produce a pH neutral salt.

There are several synthetic approaches to generating C₃ and othermalonic acid derivatives, to yield a single isomer, but most methods arenot easily scaled up to generate sufficient compound for clinicalapplications. One method which may allow scale-up of synthesis of C₃ tothe quantities needed for clinical testing and development of C₃ andlike compounds as pharmaceuticals has been developed. However,preparations of C₃ using this method have been found to include asignificant amount of a waxy contaminant which is highly toxic in cellcultures and in animals. This provides and describes methods for theremoval of contaminants from preparations of malonic acid or malonicacid/acetic acid C60 derivatives.

Exemplary protocol: A 55 gram (g) lot of C₃ was received (C-Sixty, Ltd.,Carbon Nanotechnologies Inc., Houston Tex.), who had commissionedsynthesis of a stock of C₃ from Regis Technologies (Morton Grove, Ill.).

Attempts were made to dissolve the red powder in dilute NaOH, but asignificant amount of particulate material which did not dissolve evenwith extensive mixing. When the partially solubilized solution wastested in neuronal cell cultures, it showed toxicity (increased neuronaldeath). The Regis preparation was also toxic when administered to miceat doses which had been non-toxic when internal preparations of C₃ wereused. LC-MS on the compound indicated that there was a CO₂-containingcomponent in the Regis preparation that was not present in pure C₃samples prepared by alternative synthetic approaches. Absorptionspectroscopy also indicated that there were contaminants which absorbedin the region 200-415 nm which were not present in preparations of C₃which were previously documented to be non-toxic. Finally, it wasobserved that over time, a whitish waxy material precipitated out ofsolution in the Regis preparation but not C₃.

A 5 g lot of C₃ (prepared by J-Star, South Plainfield, N.J.) using thesame template synthesis also contained the same precipitate/contaminant.

Procedure for Removal of Contaminants:

1) Dissolve powder in dilute sodium hydroxide (NaOH; range 0.25-2N), atC₃ concentrations between 10 mM-400 mM (10 mg/ml-400 mg/ml) at 4 degreesC. with stirring.

2) Add more concentrated NaOH (for example 5 N NaOH) drop-wise toachieve pH ˜7.0. The current Regis C₃ preparation has required 4.8 mEqper g to produce a solution at pH 7.0.

3) The sample is then allowed to sit at 4 degrees in the dark for 0.5-3hours.

4) The sample is centrifuged at 6000 g×30-60 minutes, which produces aclear dark red supernatant, and a solid light pink pellet. Thesupernatant is carefully removed by pipet to another tube. Thesupernatant can be allowed to sit at 4 degrees for an additional 3-4hours, and then centrifuged again to be sure that all undissolvedmaterial is removed. The pellet with insoluble waxy material containsthe contaminant, and small amounts of residual C₃, which can beextracted by additional NaOH, and repeat centrifugation.

5) The purified C₃ solution may have a minor amount of volatilecontaminant that can be further removed by vacuum distillation or bybubbling an inert gas (e.g. nitrogen, argon) through the solution.

6) An alternative approach to removing the insoluble waxy contaminantafter solubilization in dilute NaOH is to filter the sample through afilter which allows only aqueous solutions to pass.

7) Additional approaches could include 2-phase extraction if the waxycontaminant, the use of resins or other substances which can bind to thewaxy contaminant.

8) An alternative approach would be to use antibodies directed againstC60, C₃, or other malonic acid derivatives to precipitate the purecompounds away from the contaminant. Differential centrifugation oraffinity column chromatography are two potential methods to then capturethe fullerene-antibody complex.

Characterization of Purified Product from Centrifugation-BasedPurification:

1) Samples were evaluated by absorbance spectroscopy. Pure C₃ has acharacteristic spectrum with a maximum at 486 nm and a minimum at 413 nmthat allows purity and concentrations to be assessed. A max/min ratioof >4.0 is highly pure. Using absorption at 486 nm and an extinctioncoefficient of 4200 mol cm-1 the concentration to be calculated.

2) HPLC was also performed to determine the presence of other non-C₃isomers or decarboxylation products of C₃ in the purified solution.

Results:

FIG. 36 illustrates the absorption spectra of pure C₃ prepared by theBingel procedure. Purity of the sample was confirmed by HPLC, NMR, andtitration, and was >98% pure C₃. The maximum (485 nm)/minimum (415 nm)ratio is a measure of purity, with a theoretical ratio of 4.1 forcompletely pure C₃. The sample in FIG. 36 exhibits a ratio of 4.1.

FIG. 37 illustrates absorption spectra of Regis C₃ prior to clean-up.Prior to clean-up, J-Star showed similar spectrum.

FIG. 38A and FIG. 38B illustrates absorption spectrum of C₃ (Regis)after purification using the exemplary protocol (method) of thisinvention at 2 dilutions to allow all wavelengths of the spectrum to beviewed on scale. After clean-up, the max/min was 4.1

FIG. 39A and FIG. 39B illustrate neuroprotection against NMDA toxicityby a lot of pure C₃ using the exemplary purification protocol of thisinvention. Neuronal cell cultures were exposed to NMDA (150 uM) for 10minutes in the presence of different concentrations of C₃ and the amountof neuronal death assays by lactate dehydrogenase (LDH) release fromdying cells.

Contaminated C₃ shows direct toxicity on neuronal cell cultures: RegisC₃ was applied directly to neuronal cultures at the indicatedconcentrations, and cell death assayed by LDH release. Cell death wasincreased at concentrations of contaminated C₃ above 3 μM. Pure C₃ isnot toxic below 300 μM.

Example 3: Compositions and Methods Effective in the Amelioration ofInflammation and/or Oxidative Stress in the CNS Caused or Mediated byIL-6 and NADPH Oxidase

This example demonstrates that the compositions and methods of theinvention are effective to ameliorate, treat or prevent inflammationand/or oxidative stress in the CNS, e.g., brain. In alternativeembodiments, compositions and methods of the invention are used toameliorate (including to slow, reverse or abate) or prevent theincreasing vulnerability to CNS neurodegenerative disorders related topathologies, diseases (including infections) and conditions associatedwith an increased amount of CNS inflammation and/or CNS oxidativestress, including Alzheimer's disease, Lewy Body Disease, Parkinson'sDisease, Huntington's Disease, Multi-infarct dementia (vasculardementia), senile dementia. Frontotemporal Dementia (Pick's Disease) andrelated conditions.

These studies demonstrate that inflammation in the CNS (e.g., brain),acting through NADPH oxidase, constitutes a novel target for treatmentsto ameliorate, halt or reverse pathology in any individual having anyCNS neurodegenerative disorder, disease, infection, injury or conditionsassociated with an increased amount of CNS inflammation and/or CNSoxidative stress.

We tested the possibility of an increased expression of some isoforms ofthe enzyme in the aged brain. We have analyzed the presence andexpression of isoforms of Nox and tested whether Nox activitycontributes to superoxide levels in the aged brain. The mRNAs for Nox2,Nox4, and p22^(phox) were increased in several brain regions of agedmice, as illustrated in FIG. 31A), and Western blot analysis offorebrain proteins demonstrated an increase in Nox2, Nox4 and p22protein content, as illustrated in FIG. 31B. The specificity of theantibodies used for Nox2 was confirmed in gp9l phox−/− forebrainextracts, as illustrated in FIG. 31C.

FIGS. 31A-31C illustrate data showing the expression of Nox(s) in brainin young (4 mo) and old (24 mo) mice. FIG. 31A: RT-PCR depicting mRNAexpression of Nox2, Nox4 and required subunits is induced in brain ofold (24 mo) compared to young (4 mo) C57BL6 mice. Forebrains of youngand old (FIG. 31B) or wild type and gp91phox−/− (FIG. 31C) were lysed insuper-RIPA buffer and 50 μg proteins were resolved in 10% SDS-PAGE gels.Antigen recognition was assessed by Western blots using anti-gp91phoxantibodies (monoclonal 54.1 or BD-Transduction) anti-p47 (Santa Cruz),anti-p22 (monoclonal 44.1) followed by secondary antibodies conjugatedto HRP. Detection was performed using chemiluminescence (Pierce).

To further confirm the neuronal expression of Nox isoforms, we analyzedthe presence of Nox2 by fluorescence immunohistochemistry in thehippocampal region of young and old animals. The pyramidal layer of CA1had shown a substantial increase in ROS upon aging, and Nox2 was highlyexpressed in this region, as illustrated in FIGS. 32A-32B, in bothneurons and astrocytes.

FIGS. 32A-32B illustrate data showing that aging (old) mice showincreased immunostaining for Nox proteins. Immunohistochemistryperformed on brain slices from young and old animals revealed increasedNox2. Nox2 expression was increased in neurons and astrocytes in oldanimals. Confocal imaging of the neuronal marker, MAP2 (red),gp91^(phox) (green) and merged images. Antibodies were polyclonalanti-MAP2 (1:2000 Chemicon), and monoclonal 54.1 gp91^(phox) (1:300).

Confocal imaging of in vivo superoxide production showed elevated levelsof superoxide in the pyramidal layer of CA1 in the aged hippocampus,which were prevented by oral administration of the brain-permeable SODmimetic C₃, and by the Nox inhibitor apocynin, as illustrated by thedata in FIGS. 33A-33D. Since the main Nox isoforms expressed in brainare Nox2 and Nox4, and apocynin does not affect Nox4 activity, weconcluded that the source of superoxide being induced in the aged brainis Nox2.

Supporting this conclusion, Nox activity was increased in synaptosomesprepared from brains of old mice compared to young animals, asillustrated by the data in FIG. 33C, and was associated with superoxideproduction as detected by spin-trapping EPR spectroscopy. Nox enzymesare constitutively active in neurons in vivo and in synaptosomes, and itwas found that their rate of O₂ consumption was equivalent to that ofmitochondria, demonstrating that Nox is an important source ofsuperoxide at the synapse and thus contributes to age-dependent deficitsin synaptic plasticity.

The mechanisms of induction of Nox in brain are unknown, but studies inphagocytes show that inflammatory mediators are strong inducers of itsactivity. Since increased inflammatory cytokines have been described inthe aged brain, with increased levels of IL-6 being the most consistentfinding across species, we decided to study the effects of thisinterleukin in Nox induction in primary neuronal cultures and in vivo.

Exposure of cortical neurons to IL-6 (20 ng/ml, 1 h in the absence ofastrocytes) increased the phosphorylation of the protein kinase Jak2,which transduces the signal from the activated IL-6 receptor, asillustrated by the data shown in FIG. 34A. These results confirm thatthe interleukin acts directly on neurons. Prolonged exposure (24 h) tothe interleukin increased production of superoxide (as determined by DHEoxidation) and increased the expression of Nox2 in neurons, asillustrated by the data shown in FIG. 34B. The role of Nox2 activationin the increase in DHE oxidation was confirmed by co-exposure to the Noxinhibitor apocynin (0.5 mM) (FIG. 34B bottom panels).

For FIGS. 34A-34B: primary neuronal cultures were developed oncoverslips as described Kinney et al., 2006, supra. After 21 days inculture, coverslips containing neurons were separated from theastrocytic monolayers, washed in HCSS and subjected to IL-6 treatmentfor 1 hour (FIG. 34A) or treated with IL-6 for 24 h on top of theastrocyte monolayer (FIG. 34B). After treatment, the coverslips werefixed in paraformaldehyde and processed for double fluorescenceimmuno-cytochemistry using the following antibodies: anti-GAD67 (1:2000,Chemicon. Red) anti-phospho-Jak2 (1:100, Cell Signaling. Green), andanti-Nox2 (1:300, monoclonal 54.1 Green). For detection of ROS, DHE (1μg/ml. Red) was applied for the last hour of treatment. Images wereobtained using a Zeiss confocal microscope with a 40× water immersionobjective. Secondary antibodies were conjugated to ALEXAFLUOR 488™(green fluorescence) and ALEXAFLUOR 568™ (red fluorescence).

FIG. 35 illustrates that IL-6 treatment in vivo increases Nox2 mRNA inbrain, as well as Nox protein and activity in synaptosomes. Four monthold C57Bl/6 were treated with either saline (saline or control) or withIL-6 (5 μg/kg) on two consecutive days, and brains were either processedfor RNA (FIG. 35A) or for synaptosomal preparation (FIG. 35B and FIG.35C). Nox2 mRNA was detected by RT-PCR as described herein. Fordetection of Nox2 and p22, antibodies against the corresponding proteinswere used on immunoblots of 50 μg of synaptosomal proteins separated on10% SDS-PAGE gels. Antibodies used were anti-Nox2 (54.1, 1:1000), antip22 (44.1: 1:500), and anti-actin (1:30,000, Chemicon). Synaptosomal Noxactivity was assayed as described above.

Example 4: Compositions and Methods of the Invention are Effective inthe Amelioration of Aging and Frailty Syndrome (FS)

This example demonstrates that the compositions and methods of theinvention are effective to ameliorate, treat or prevent frailty syndrome(FS), and the CNS neurodegenerative, cognitive, learning or memoryimpairments resulting therefrom. FS is a recognized condition seenparticularly in older patients characterized by, e.g., low functionalreserve, easy tiring, decrease of libido, mood disturbance, acceleratedosteoporosis, decreased muscle strength, and high susceptibility todisease. This example demonstrates that eliminating IL-6 appears toblock features of the frailty syndrome, and that this may be mediated byreducing superoxide levels.

Age-related reduction in number of parvalbumin-interneurons in theprefrontal cortex is demonstrated by data shown in FIGS. 40A-40C. Theprefrontal cortex (including the pre-limbic and infra-limbic regions)was analyzed for the expression of the calcium binding proteins (CBP)parvalbumin (PV), calbindin (CB), and calretinin (CR). Fluorescentstaining for these markers showed a different distribution for each CBP,as shown in FIG. 40A. Analysis of the number of cells expressing eachCBP was performed across 6 consecutive coronal sections comprising theregions between Bregma 2.0 and 1.3, as shown in FIG. 40B, and thecumulative results for the expression of each CBP are shown in FIG. 40C.*=statistically significant (P<0.001) with respect to young by one-wayANOVA followed by Tukey's test. N=6 animals per condition.

Age-related decrease of PV-interneurons in prefrontal and hippocampalregions: long-term chronic treatment with an SOD-mimetic preventsinterneuron loss is demonstrated by data shown in FIGS. 41A-41B. Coronalbrain slices of young (YM) and old (OM) male mice were stained forparvalbumin and total PV-positive cell counts were evaluated across 4slices of the prelimbic region (PFC) and hippocampal regions CA1, CA3and dentate gyrus (DG), as shown in FIG. 41A, and as described in detailin Example 3, above. Aging was accompanied by a statisticallysignificant decrease in PV-interneuron number in all regions analyzed,as shown in FIG. 41B. A reduction of 17.1±6.8% (p=0.008) was observed inPFC, and of 45.1±17.7% in area CA3 (p=0.002), which was the mostpronounced decrease of all regions analyzed. Treatment of animals frommiddle age with the SOD-mimetic C3 (OM+C3) prevented the reduction ofPV-interneuron numbers in CA1 and CA3, but not in DG as shown in FIG.41C. Statistical significance was determined by ANOVA followed byTukey's test. YM and OM: n=9 animals per group; OM+C3: n=7 animals.

The aged prefrontal cortex is more vulnerable to the effects of ketamineon parvalbumin and calbindin interneurons, as demonstrated by data shownin FIGS. 42A-42C. Brain coronal sections (50 mm) from animals (young andold) treated with saline or ketamine (15 mg/kg, i.p.) were doublestained for each CBP and GAD67. The median fluorescence intensity percell was obtained for each section and averaged across all sections ofthe animal to obtain the mean intensity per cell across the PFC of eachanimal. Results obtained for all animals were normalized by the averagemean intensity per cell obtained for the saline treated controls andexpressed as percentages of control conditions. FIG. 42A: Effect ofketamine on the average mean intensity per cell for each CBP in the PFCregion. FIG. 42B: Analysis of the mean intensity per cell for GAD67content analyzed in each CBP stained cell. FIG. 42C: Confocal imagesobtained with a 40× objective depicting the effects of ketamine on theimmunofluorescence for PV and GAD67 in the PFC region of young and oldanimals (Bar=20 mm). *=statistically significant with respect to salinecontrol (CB: P<0.001; GAD67 in CB cells: P=0.024; PV: P<0.001; GAD67 inPV cells: P=0.003) by ANOVA followed by Tukey's test.

Aging increases the vulnerability to Nox-dependent loss of phenotype ofPV-interneurons and sensitivity to low doses of an anesthetic(ketamine), as illustrated by data shown in FIGS. 43A-43B. As shown inFIG. 43A, in addition to loss of PV-interneurons with aging, there isenhanced vulnerability of the remaining neurons to loss-of-phenotype(and loss of inhibitory function) in old mice in response to evensub-anesthetic doses of an anesthetic, in this case, ketamine (15 mg/kgi.p. on two consecutive days). Aging also increases the sensitivity toketamine, demonstrated using a “loss of righting reflex (LORR) test, asshown in FIG. 43B, with significantly greater loss-of-righting reflex inold (versus young) animals at the same dose of ketamine.

Plasma IL-6 is increased with aging or after intraperitoneal (i.p.)administration of IL-6, as illustrated by data shown in FIG. 44. Plasmalevels of IL-6 are significantly increased in 24-month old C57B6 miceversus young (4 month) mice. IL-6 was assayed by ELISA (R&D Systems,Minneapolis, Minn.). Mice were then given a direct intraperitoneal (IP)injection of 3 μg/kg IL-6 on two consecutive days, and plasma IL-6 wasassayed 16 hours (hr) after the last injection. IL-6 injection increasedplasma IL-6 in young, but not old, mice. Since the half-life of IL-6 inplasma is 4 hr. the sustained increase in IL-6 after injection mayindicate induction of new IL-6 synthesis in young mice, which may besuppressed by the high endogenous IL-6 levels in old mice. Higher dosesof IL-6 (12 μg/kg) induced the sickness response and generalizedinflammatory reaction, so 3 μg/kg/day for two consecutive days was usedfor all subsequent studies.

NFkB (p65) activity was measured in brain nuclear extracts from oldwild-type (WT) (“CTL”, or control) versus old IL-6−/− mice (“IL-6 KO”,or IL-6 knockout) by an ELISA kit for the p65 subunit of NFkB, with “nooligo” and “mutant oligo” controls, as illustrated by data shown in FIG.45. Old IL-6−/− mice have significantly lower NFkB activity than old WTmice. Since NFkB regulates expression of Nox subunits including Nox2,p22phox and p47phox, among others, decreased NFkB activity in IL-6−/−mice would reduce expression of Nox isoforms in aging, as observed.

RNA expression of IL-1b and TNFa was measured in brain extracts from oldwild-type and old IL-6−/− mice (as in FIG. 45), as illustrated by datashown in FIG. 46, indicating that lack of IL-6 expression in the IL-6−/−mice does not modify expression of IL-1β or TNFα. Lane with X did nothave RNA loaded. GAPDH RNA expression serves as internal control.

Nox-dependent superoxide production is lower in synaptosomes from IL-6deficient (IL-6-KO) old mice compared to age-matched (old) wild-typecontrols, as measured by EPR, with spectra illustrated in FIG. 47, left,as graphically illustrated in FIG. 47, right; demonstrating that IL-6increases Nox-dependent superoxide production.

Performance of IL-6 deficient (IL-6-KO) old mice compared to age-matched(old) wild-type controls on a rotorod test showed that in day 2 and day3 test samples the presence of IL-6 decreased the level of performance,as illustrated by data shown in FIG. 48. The rotorod is designed toassess motor coordination, balance and equilibrium. The mouse can beplaced on a rod and the rotorod accelerates gradually. Latencies for themice to fall from the rod are recorded. A rotorod can be a semi-enclosedchamber which contains a beam made of ribbed plastic and flanked byround plates on either side to prevent any escape (e.g., AccuscanInstruments, Columbus, Ohio). The rod can be suspended at a height(e.g., 35 cm) above the floor. The mouse is placed on top of the beamfacing away from the experimenter's view, in the orientation opposite tothat of its rotation, so that forward locomotion is necessary for fallavoidance. The rotorod can be accelerated gradually without jerks from 0to 35 rpm over a 2-minute trial. Latencies for the mice to fall from therod can be recorded automatically by a computer. Each mouse can be given2 to 5 trials with a 15-min inter-trial interval on each of 3consecutive days.

IL-6 knockout (IL-6−/−) male mice retain reproductive fecundity intolate-life compared as to wild-type controls; thus, the presence of IL-6decreases fecundity in late-life. The number of litters and pupsfathered was recorded for 4 IL-6−/− mice versus more than 10 controlmales. Six of the litters were fathered by IL-6−/− mice older than 20months of age. No litters were fathered by WT mice past 14 mos of age.

Genotype (C57BL6 # of litters fathered background) Gender n after 14 moof age # of pups WT M >10 0 0 IL-6(−/−) M 4 13 70 Oldest fathered 24mos+

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

What is claimed is:
 1. A method of: decreasing superoxide and/orhydrogen peroxide levels in a cell, or, inhibiting or decreasing thelevel of activation or activity of an NFkB, an IL-6, an IL-6-R, or anNADPH oxidase, comprising (a) providing a C60 malonic acid derivative;and (b) administering the C60 malonic acid derivative to the cell,wherein optionally the administering is in vitro or in vivo.
 2. Themethod of claim 1, wherein administering the C60 malonic acid derivativein vivo to an individual in need thereof treats, ameliorates, slows theprogress of or reverses: a schizophrenia; a psychosis; a delirium,optionally a post-operative delirium; a drug-induced psychosis or apsychotic feature; frailty syndrome (FS), a cognitive impairment, or alearning or memory impairment, resulting from or associated with frailtysyndrome (FS), aging, depression, dementia; a traumatic war neurosis; apost traumatic stress disorder (PTSD) or a post-traumatic stresssyndrome (PTSS); Amyotrophic Lateral Sclerosis (ALS, or Lou Gehrig'sDisease); Multiple Sclerosis (MS); a cognitive, learning or memoryimpairment; a CNS inflammation, optionally a CNS inflammation resultingfrom a trauma or an inflammation, optionally an inflammation from a CNSinfection; Alzheimer's disease; Lewy Body Disease; Parkinson's Disease;Huntington's Disease; a dementia, optionally a multi-infarct dementia(vascular dementia) or a senile dementia; or, Frontotemporal Dementia(Pick's Disease).
 3. The method of claim 1, wherein administering theC60 malonic acid derivative in vivo to an individual in need thereof:treats, ameliorates, slows the progress of, abates, prevents orreverses: neuron damage, CNS damage or brain damage in an individualhaving frailty syndrome (FS), aging, an injury, a pathology, a disease,an infection, a condition causing and/or associated with an increasedamount of CNS inflammation and/or CNS oxidative stress; or acceleratingthe recovery of CNS neuron or brain damage in individuals having aninjury, a pathology, an Amyotrophic Lateral Sclerosis (ALS, or LouGehrig's Disease), Multiple Sclerosis (MS)), and/or cognitive, learningor memory impairments resulting therefrom, a disease, an infectionand/or a condition causing and/or associated with an increased amount ofCNS inflammation and/or CNS oxidative stress, and/or cognitive, learningor memory impairments resulting therefrom.
 4. The method of claim 1,wherein the C60 malonic acid derivative is formulated as apharmaceutical formulation, and optionally the pharmaceuticalformulation is formulated for delivery to the CNS, or brain or a CNSneural cell, or for passing through the blood brain barrier (BBB). 5.The method of claim 1, wherein the C60 malonic acid derivative isformulated for delivery to a parvalbum in-positive GABA-ergicinterneuron.
 6. The method of claim 1, further comprising administrationof a small molecule, wherein optionally the small molecule comprises ano-methoxycatechol, an apocynin, a diapocynin,4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF),4-hydroxy-3′-methoxy-acetophenon, N-Vanillylnonanamide, staurosporine ora combination thereof.
 7. A method of protecting the function of, ormaintaining the level of activation or activity of cortical inhibitoryinhibitory neurons, or parvalbum in-positive GABA-ergic interneuronscomprising: contacting the cortical inhibitory inhibitory neurons, orparvalbumin-positive GABA-ergic interneurons in vitro with a compositioncomprising a superoxide dismutase mimetic that is capable of decreasingsuperoxide and/or hydrogen peroxide levels, wherein the superoxidedismutase mimetic comprises a C60 fullerene, C3 tris malonic acid C60fullerene or a malonic acid derivative.
 8. The method of claim 1,wherein the C60 malonic acid (propanedioic acid) derivative is purifiedby a method comprising: (i) (a) dissolving an impure powder form of aC60 malonic acid derivative in a dilute sodium hydroxide (NaOH) solutionat a concentration of between about 1 mM to 400 mM at about 4 degrees C.with stirring, wherein optionally the C60 malonic acid derivative is orcomprises a C3 tris malonic acid C60 fullerene or a C60 macrocyclicmalonate derivative, and optionally the malonate is functionalized witha halide atom, or malonate ester groups are replaced by alkyne groups,optionally as a dialkynylmethanofullerene; (b) adding a second solutionof NaOH more concentrated than the dilute NaOH solution in step (a)drop-wise to the solution of step (a) to achieve an approximatelyneutral pH; (c) incubating the solution of step (b) at 4 degrees C. inthe dark for approximately 0.5 to 3 hours; (d) centrifuging the solutionafter the incubating of step (c) to produce a clear dark red supernatantand a solid light pink pellet; (e) removing the supernatant to adifferent container; (f) incubating the supernatant removed in step (e)at 4 degrees C. for an additional about 3 to 4 hours; and (g) (1)re-centrifuging to remove substantially all or all undissolved materialto generate a pellet and a solution comprising purified C3 tris malonicacid C60 fullerene, wherein the pellet comprises an insoluble waxymaterial containing contaminant and small amounts of residual C3 trismalonic acid C60 fullerene, or (2) filtering the sample through a filterwhich allows only aqueous solutions to pass, thereby removing aninsoluble waxy contaminant after solubilization in dilute NaOH, therebygenerating a solution comprising purified C3 tris malonic acid C60fullerene; or (ii) the method of (i), wherein the purified C3 solutionis further treated to remove a minor amount of volatile contaminant byvacuum distillation or by bubbling an inert gas through the solution. 9.The method of claim 1, wherein the C60 malonic acid (propanedioic acid)derivative is purified by a method comprising: (i) (a) providing asolution comprising an impure powder form of a C60 malonic acidderivative, wherein optionally the C60 malonic acid derivative is orcomprises a C₃ tris malonic acid C60 fullerene or a C60 macrocyclicmalonate derivative, and optionally the malonate is functionalized witha halide atom, or malonate ester groups are replaced by alkyne groups,optionally as a dialkynylmethanofullerene; (b) providing an antibodydirected against a C60 fullerene or a C60 malonic acid derivative,wherein optionally the C60 malonic acid derivative is a C₃ tris malonicacid C60 fullerene; and (c) isolating the C60 fullerene or the C60malonic acid derivative by incubating the antibody with the C60fullerene or the C60 malonic acid derivative under conditions whereinthe antibody specifically binds to the C60 fullerene, or the C60 malonicacid derivative; or (ii) the method of (i), wherein an antibody-C60fullerene complex or an antibody-C₃ tris malonic acid C60 fullerene orantibody-malonic acid derivative complex is purified by gelelectrophoresis purification, HPLC, immunoprecipitation, columnchromatography, differential centrifugation or affinity columnchromatography.
 10. The method of claim 1, wherein the C60 malonic acidderivative is or comprises a C3 tris malonic acid C60 fullerene or a C60macrocyclic malonate derivative.
 11. The method of claim 1, wherein themalonate derivative is functionalized with a halide atom, or malonateester groups are replaced by alkyne groups, and optionally the malonatederivative is dialkynylmethanofullerene.
 12. The method of claim 7,wherein the malonic acid derivative is or comprises a macrocyclicmalonate compound.
 13. The method of claim 7, wherein the malonate isfunctionalized with a halide atom, or malonate ester groups are replacedby alkyne groups, and optionally the malonate derivative isdialkynylmethanofullerene.
 14. The method of claim 2, whereinadministering the C60 malonic acid derivative in vivo to an individualin need thereof treats, ameliorates, slows the progress of or reverses:a schizophrenia; a psychosis; a delirium, optionally a post-operativedelirium; a drug-induced psychosis or a psychotic feature; frailtysyndrome (FS), a cognitive impairment, or a learning or memoryimpairment, or a learning or memory impairment resulting from orassociated with frailty syndrome (FS).
 15. The method of claim 2,wherein administering the C60 malonic acid derivative in vivo to anindividual in need thereof treats, ameliorates, slows the progress of orreverses aging.
 16. The method of claim 2, wherein administering the C60malonic acid derivative in vivo to an individual in need thereof treats,ameliorates, slows the progress of or reverses depression.
 17. Themethod of claim 2, wherein administering the C60 malonic acid derivativein vivo to an individual in need thereof treats, ameliorates, slows theprogress of or reverses dementia, and optionally the dementia is amulti-infarct dementia (vascular dementia) or a senile dementia; or, aFrontotemporal Dementia (Pick's Disease).
 18. The method of claim 2,wherein administering the C60 malonic acid derivative in vivo to anindividual in need thereof treats, ameliorates, slows the progress of orreverses: a trauma, a traumatic war neurosis; a post traumatic stressdisorder (PTSD) or a post-traumatic stress syndrome (PTSS).
 19. Themethod of claim 2, wherein administering the C60 malonic acid derivativein vivo to an individual in need thereof treats, ameliorates, slows theprogress of or reverses Amyotrophic Lateral Sclerosis (ALS, or LouGehrig's Disease); Multiple Sclerosis (MS).
 20. The method of claim 2,wherein administering the C60 malonic acid derivative in vivo to anindividual in need thereof treats, ameliorates, slows the progress of orreverses: a cognitive, learning or memory impairment; a CNSinflammation, optionally a CNS inflammation resulting from aninflammation, optionally an inflammation from a CNS infection;Alzheimer's disease; Lewy Body Disease; Parkinson's Disease;Huntington's Disease.